EP1385877B1 - Tapp2 polypeptide - Google Patents

Tapp2 polypeptide Download PDF

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EP1385877B1
EP1385877B1 EP01954140A EP01954140A EP1385877B1 EP 1385877 B1 EP1385877 B1 EP 1385877B1 EP 01954140 A EP01954140 A EP 01954140A EP 01954140 A EP01954140 A EP 01954140A EP 1385877 B1 EP1385877 B1 EP 1385877B1
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polypeptide
ptdins
binding
domain
protein
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EP1385877A2 (de
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Simon Medical Research Council DOWLER
David Medical Research Council CAMPBELL
Alexander Gray
Peter Downes
Dario Med Resch Counc.Prot. Phospho.Unit ALESSI
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Medical Research Council
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Medical Research Council
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to polypeptides, polynucleotides and uses thereof, in particular to polypeptides comprising a PH (pleckstrin homology) domain.
  • PtdIns(4,5)P 2 phosphoinositide 3-kinase family which phosphorylate phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2 ) at the D-3 position of the inositol ring to generate the lipid second messenger, PtdIns(3,4,5)P 3 [1].
  • PtdIns(3,4,5)P 3 A group of proteins has been identified that possess a certain type of pleckstrin homology (PH) domain which interacts specifically with PtdIns(3,4,5)P 3 and often its immediate breakdown product, PtdIns(3,4)P 2 , also thought to be a signalling lipid (reviewed in Lemmon & Fergusson (2000) Biochem J 350, 1-18 ).
  • PH pleckstrin homology
  • proteins that bind specifically to a phosphoinositide other than PtdIns(3,4,5)P 3 in particular PtdIns3P, PtdIns3,4P 2 or PtdIns4P.
  • the proteins each possess a PH domain which is considered to contain a PPBM and which binds the said phosphoinositide but not to PtdIns(3,4,5)P 3 .
  • These proteins may play important roles in triggering cellular processes that are regulated by other phosphoinositides.
  • the proteins/PH domains may be useful in drug screening assays, in particular for compounds that may be useful in treating cancer, diabetes or stroke. They may also be useful in measuring concentrations and/or locations of the phosphoinositide lipids PtdIns3P, PtdIns3,4P 2 and PtdIns4P.
  • An aspect of the invention provides the use of a polypeptide capable of binding specifically to PtdIns(3,4)P 2 , but not capable of binding to PtdIns(3,4,5)P 3 , in as screening method for identifying a compound suitable for modulating signalling by PtdIns(3,4)P 2 , wherein the polypeptide comprises a PH domain capable of binding to PtdIns(3,4)P 2 but not capable of binding to PtdIns(3,4,5)P 3 wherein the polypeptide may comprise the amino acid sequence or or a fragment thereof, said fragment being capable of binding specifically to PtdIns(3,4)P 2 but not capable of binding to PtdIns(3,4,5)P 3 , or the polypeptide may comprise a fusion of a said fragment thereof.
  • Polypeptides capable of binding to PtdIns(3,4)P 2 , but not capable of binding to PtdIns(3,4,5)P 3 have not previously been identified as such. Screening methods making use of such a polypeptide have not previously been proposed.
  • Plecktrin Homology (PH) domain is well known to those skilled in the art. These domains or ⁇ 100 residues are found in over 70 other proteins and are predicted to fold into a similar 3-dimensional structures and may mediate protein-lipid, protein-protein interactions, or both ( Gibson, T.J. et al (1994) Trends Biochem. Sci. 19, 349-353 ; Shaw, G. (1996) Bioessays 18, 35-46 ). Polypeptides with PH domains of determined tertiary sructure include plecktrin, spectrin, dynamin, and phospholipase C- ⁇ . Although the percentage identity is poor between PH domains in general there are certain positions that show high levels of residue type conservation.
  • P utative P tdIns(3,4,5)P3 B inding M otif is known to those skilled in the art.
  • the motif is K-X-Sm-X 6-11 -R/K-X-R-Hyd-Hyd motif (where X is any amino acid, Sm is a small, preferably uncharged, amino acid and Hyd is a hydrophobic amino acid) and lies near the N-terminal end of the PH domain.
  • a small amino acid is included glycine, alanine, threonine and serine.
  • An aspartate or proline amino acid residue may alternatively be present at the position in the motif where a small amino acid is preferred.
  • hydrophobic amino acid By a hydrophobic amino acid is meant tyrosine, leucine, isoleucine, tryptophan and phenylalanine.
  • a glutamine amino acid residue (for example) may alternatively be present at the first position where a hydrophobic amino acid residue is preferred.
  • a glutamine, asparagine or histidine amino acid residue may be present at a position where a lysine or arginine residue is preferred. It is strongly preferred that an acidic or hydrophobic residue is not present at a position where a lysine or arginine residue is preferred, or at the position in the motif where a small amino acid is preferred. It is preferred that the PH domain has at least five of the six specified residues of the PPBM.
  • the PH domain has both hydrophobic amino acids of the motif and/or the first lysine (K) residue of the motif. It is preferred that the PH domain also has a tryptophan residue at the position equivalent to position 280 of TAPP1, as discussed above.
  • the said polypeptide binds specifically to PtdIns(3,4)P 2 , ie is able to bind to PtdIns(3,4)P 2 , and is substantially unable to bind to other phosphoinositides, in particular PtdIns(3,4,5)P 3 .
  • able to bind binding of the said polypeptide to the said phosphoinositide can be detected using a surface plasmon resonance or protein lipid overlay technique as described in Example 1 and the legends to Table 2 and Figure 4 .
  • substantially unable to bind binding of the said polypeptide to the said phosphoinositide is not detected, or is only weakly detected using a surface plasmon resonance or protein lipid overlay technique as described in Example 1 and the legends to Table 2 and Figure 4 .
  • the polypeptide binds to PtdIns(3,4)P 2 , with at least two, preferably 3, 5, 10, 15, 20, 30 or 50-fold higher affinity than to other phosphoinositides, in particular PtdIns5P, PtdIns(4,5)P 2 , PtdIns(3,4,5)P 3 , PtdIns3P, PtdIns4P and PtdIns(3,5)P 2 .
  • the binding of the said polypeptide to PtdIns(3,4)P 2 has an apparent K D of less than about 2000 nM, 1000 nM or 500 nM, preferably less than about 400 or 350 nM, for example between about 350 nM and 10 nM, when measured using the method described in Example 1.
  • the binding of the said polypeptide to other phosphoinositides has an apparent K D of more than about 2000 nM, 1000 nM or 500 nM when measured using the method described in Example 1.
  • polypeptides that bind specifically to PtdIns(3,4)P 2 are considered to include mammalian (for example human and mouse) TAPP (for example TAPP1 and TAPP2), and fragments and fusions thereof that comprise the C-terminal PH domain, as discussed further below and in Example 1. Further examples are considered to include fragments, variants, Table 2: Apparent K d of PEPP1, FAPP1 wild type and mutant TAPP1 and TAPP2 for binding to phosphoinositides as measured by surface plasmon resonance. The binding of the indicated GST-fusion proteins phosphoinositides incorporated into supported phosphatidylcholine monolayers was measured as described in the experimental section.
  • the affinities were determined by global fitting of the association and dissociation curves to a 1:1 binding model. Abbreviations used, FL full length protein; NT-PH, N-terminal PH domain; CT-PH, C-terminal PH domain; NB, no binding detected; ND, not determined.
  • the binding of the indicated GST-fusion proteins phosphoinositides incorporated into supported phosphatidylcholine monolayers was measured as described in the experimental section.
  • the apparent affinities were determined by global fitting of the association and dissociation curves to a 1:1 binding modeland were used to rank the binding affinity relative to that of TAPP1 to PtdIns(3,4)P 2 which was approximately 5 nM.
  • Abbreviations used FL full length protein; NT-PH, N-terminal PH domain; CT-PH, C-terminal PH domain; NB, no binding detected; ND, not determined.
  • polypeptides that bind specifically to PtdIns4P are considered to include FAPP, for example mammalian FAPP (for example human or mouse FAPP) or Xenopus or Zebrafish FAPP, for example human FAPP1 or FAPP2 and fragments and fusions thereof that comprise a PH domain, as discussed further below and in Example 1. Further examples are considered to include fragments, variants, derivatives or fusions thereof, or fusions of fragments, variants or derivatives, that retain the said phosphoinositide binding properties, as discussed further below.
  • FAPP for example mammalian FAPP (for example human or mouse FAPP) or Xenopus or Zebrafish FAPP, for example human FAPP1 or FAPP2 and fragments and fusions thereof that comprise a PH domain, as discussed further below and in Example 1.
  • polypeptides that bind specifically to PtdIns3P are considered to include mammalian (for example human and mouse) PEPP (for example PEPP1, PEPP2 and PEPP3) and plant (for example Arabidopsis ) AtPH1, and fragments and fusions thereof that comprise a PH domain, as discussed further below and in Example 1. Further examples are considered to include fragments, variants, derivatives or fusions thereof, or fusions of fragments, variants or derivatives, that retain the said phosphoinositide binding properties, as discussed further below.
  • mammalian for example human and mouse
  • PEPP for example PEPP1, PEPP2 and PEPP3
  • plant for example Arabidopsis
  • polypeptides that bind specifically to PtdIns(3,5)P 2 are considered to include centaurin- ⁇ 2 (for example mammalian, for example human or mouse, or Drosophila or C . elegans ), and fragments and fusions thereof that comprise the C-terminal PH domain, as discussed further below and in Example 1. Further examples are considered to include fragments, variants, derivatives or fusions thereof, or fusions of fragments, variants or derivatives, that retain the said phosphoinositide binding properties, as discussed further below.
  • TAPP, PEPP, FAPP, AtPH1 and centaurin- ⁇ 2 are discussed in Example 1, for example in the section relation to cloning of PH domains and in Figure 1 .
  • the screaning method of the invention comprises the steps of (1) exposing the said polypeptide to PtdIns(3,4)P 2 , in the presence of a test compound; (2) determining whether the test compound modulates binding of PtdIns(3,4)P 2 to the said polypeptide; and (3) selecting a compound which modulates binding of PtdIns(3,4)P 2 to the said polypeptide.
  • a further aspect of the invention provides a method of identifying a compound that modulates the phospholipid binding activity of a polypeptide capable of binding to PtdIns(3,4)P 2 , but not capable of binding to PtdIns(3,4,5)P 3 , according to the preceding aspect the method comprising contacting a compound with the said polypeptide and determining whether the phospholipid binding activity of the said polypeptide is changed in the presence of the compound from that in the absence of said compound.
  • Example 1 The binding of polypeptides comprising a PH domain having the required properties to phospholipids is described in Example 1. It is preferred that modulation of the binding to PtdIns(3,4)P 2 , is measured.
  • Methods of detecting binding of the said polypeptide to phospholipids are described in Example 1 and include a protein-lipid overlay assay in which the lipid is spotted onto a support, for example Hybond-C extra membrane, and protein bound to the support by virtue of interaction with the lipid is detected, for example using an antibody-based method, as well know to those skilled in the art.
  • a surface plasmon resonance assay for example as described in Example 1 or in Plant et al (1995) Analyt Biochem 226(2), 342-348 , may alternatively be used.
  • Methods may make use of a said polypeptide comprising a PH domain, or fragment thereof, that is labelled, for example with a radioactive or fluorescent labeL Suitable methods may also be described in, for example, Shirai et al (1998) Biochim Biophys Acta 1402(3), 292-302 (use of an affinity column prepared using phosphatidylinositol analogues) and Rao et al (1999) J Biol Chem 274, 37893-37900 (use of avidin-coated beads bound to biotinylated phosphatidylinositol analogues).
  • reagents including any fragment of the polypeptide
  • conditions used in the method may be chosen such that the interactions between the said polypeptide and a phosphoinositide, for example PtdIns(3,4)P 2 , are substantially the same as between the wild-type, preferably human polypeptide (for example TAPP) and the phosphoinositide or interacting polypeptide in vivo .
  • the compound decreases the relevant binding activity of said polypeptide.
  • the compound may bind substantially reversibly or substantially irreversibly to the relevant binding site of said polypeptide.
  • the compound may bind to a portion of said polypeptide that is not the binding site so as to interfere with the binding of the said phosphoinositide-binding polypeptide to the phosphoinositide.
  • the compound may bind to a portion of said polypeptide so as to decrease said polypeptide's binding activity by an allosteric effect. This allosteric effect may be an allosteric effect that is involved in the natural regulation of the said polypeptide's activity.
  • the compound may, for example, change the configuration of the polypeptide so that it is substantially unable to bind to the particular phosphoinositide.
  • the compound may be capable of affecting the intracellular location of the polypeptide; for example, it may inhibit or promote the translocation of the polypeptide to a membrane, for example the plasma membrane or golgi, vacuole, lysosome or endosome membrane. Possible association with cellular membranes of polypeptides comprising a PH domain with the required phosphoinositide binding properties are discussed further in Example 1.
  • the compound may modulate any interaction of the polypeptide with further identical polypeptide molecules (ie self-association, for example dimerisation).
  • a compound that, for example, is capable of modulating the phosphorylation or other post-translational modification of the polypeptide may thereby, for example, modulate the ability of the polypeptide to bind to a phosphoinositide.
  • a compound that is capable of modulating the ability of the polypeptide to bind to a phosphoinositide may thereby modulate the intracellular location of the polypeptide molecule and/or modulate any post-translational modification, for example phosphorylation, of the polypeptide.
  • the compound increases the binding activity of said polypeptide.
  • the compound may bind to a portion of said polypeptide that is not the relevant binding site so as to aid the binding of the said polypeptide to the phospholipid, as appropriate.
  • the compound may bind to a portion of said polypeptide so as to increase said polypeptide's binding activity by an allosteric effect.
  • This allosteric effect may be an allosteric effect that is involved in the natural regulation of the said polypeptide's activity.
  • An example of a compound that may be capable of inhibiting binding of a phosphoinositide to a said polypeptide is InsP 4 , the head group of PtdIns(3,4,5)P 3 .
  • Ins(1,3,4)P 3 , the head group of PtdIns(1,3,4)P 3 may be capable of inhibiting binding of PtdIns(3,4)P 2 to TAPP.
  • Example 1 the appropriate methods make use of the methods described in Example 1 for detecting and/or quantifying the interaction between a polypeptide and a phospholipid, for example a protein-lipid overlay or surface plasmon resonance method, as discussed above. It is preferred that a GST-tagged fusion of the polypeptide of the invention or a fragment therof is used. Methods in which radioactively or fluorescently labelled lipids are used may also be useful.
  • Suitable methods include yeast two-hybrid interactions, co-purification, ELISA, co-immunoprecipitation methods and cellular response assays.
  • Cellular response assays may be carried out in a variety of cell types, for example in adipocytes or adipocyte cell lines, in a skeletal muscle cell line (such as the L6 myotubule cell line), liver cells or liver cell lines or cancer cells or cancer cell lines.
  • Platelets may be preferred when the polypeptide is TAPP.
  • NIH Swiss mouse embryo cells NIH/3T3 available from the American Type Culture Collection (ATCC) of Rockville, MD, USA (ATCC) as CRL 1658
  • human embryonic kidney 293 cells also available from the ATCC
  • the method may be performed in vitro, either in intact cells or tissues, with broken cell or tissue preparations or at least partially purified components. Alternatively, they may be performed in vivo .
  • the cells tissues or organisms in/on which the method is performed may be transgenic. In particular they may be transgenic for the said polypeptide capable of binding a specific phosphoinositide.
  • FRET Fluorescence Energy Resonance Transfer
  • RNA transcribed from a promoter regulated by the polypeptide may be measured.
  • the protein may be one that is physiologically regulated by the polypeptide or may be a "reporter" protein, as well known to those skilled in the art (ie a recombinant construct may be used).
  • a reporter protein may be one whose activity may easily be assayed, for example ( ⁇ -galactosidase, chloramphenicol acetyltransferase or luciferase (see, for example, Tan et al (1996)).
  • the reporter gene may be fatal to the cells, or alternatively may allow cells to survive under otherwise fatal conditions. Cell survival can then be measured, for example using colorimetric assays for mitochondrial activity, such as reduction of WST-1 (Boehringer).
  • WST-1 is a formosan dye that undergoes a change in absorbance on receiving electrons via succinate dehydrogenase.
  • Promoters whose activity may be regulated by a signalling pathway in which the polypeptide may be involved may be identified using microarray technology, as known to those skilled in the art, in which the expression of multiple genes may be examined simultaneously, for example in stimulated and unstimulated cells expressing the wild-type polypeptide or a dominant negative mutation. Differences in expression patterns between the different cells/activation states indicate genes/promoters which the polypeptide may regulate.
  • An example of a dominant negative mutant of TAPP is a fragment of TAPP comrpising the C-terminal PH domain, but not the N-terminal PH domain and/or putative SH3 binding domain (TAPP2) and/or PDZ binding sequence.
  • TAPP2 putative SH3 binding domain
  • Insulin exerts important effects on gene expression in multiple tissues ( O'Brien, R. M. & Granner, D. K (1996) Physiol. Rev. 76, 1109-1161 ).
  • insulin suppresses the expression of a number of genes which contain a conserved insulin response sequence (IRS) 1 (CAAAAC/TAA), including insulin-like growth factor binding protein-1 (IGFBP-1), apolipoprotein CIII (apoCIII), phosphoenol-pyruvate carboxykinase (PEPCK) and glucose-6 phosphatase (G6Pase) ( Goswami, R et al (1994) Endocrinol.
  • IGFBP-1 insulin-like growth factor binding protein-1
  • apoCIII apolipoprotein CIII
  • PEPCK phosphoenol-pyruvate carboxykinase
  • G6Pase glucose-6 phosphatase
  • transcription of these genes may be assessed or promoters from these genes may be used in a reporter construct as described above, for example when the polypeptide is TAPP.
  • Microarray technology may be used in assessing transcription of genes or reporter constructs, as known to those skilled in the art.
  • the transcription of a gene indicated above may be measured by measurement of changes in the enzymatic or other activity of the said gene product, for example in a cell. Suitable methods will be well known to those skilled in the art.
  • the polypeptide may be cleaved, for example using trypsin, cyanogen bromide, V8 protease formic acid, or another specific cleavage reagent.
  • the digest may be chromatographed on a Vydac C18 column or subjected to SDS-PAGE to resolve the peptides.
  • the N-terminal sequence of the peptides may then be determined using standard methods.
  • sequences are used to isolate a nucleic acid encoding the peptide sequences using standard PCR-based strategies.
  • Degenerate oligonucleotide mixtures each comprising a mixture of all possible sequences encoding a part of the peptide sequences, are designed and used as PCR primers or probes for hybridisation analysis of PCR products after Southern blotting.
  • mRNA prepared from cells in which the polypeptide may be expressed is used as the template for reverse transcriptase, to prepare cDNA, which is then used as the template for the PCR reactions.
  • Positive PCR fragments are subcloned and used to screen cDNA libraries to isolate a full length clone for the polypeptide.
  • sequences of initial subcloned PCR fragments may be determined, and the sequence may then be extended by known PCR-based techniques to obtain a full length sequence.
  • the initial PCR sequence may be used to screen electronic databases of expressed sequence tags (ESTs) or other known sequences.
  • ESTs expressed sequence tags
  • related sequences may be identified which may be useful in isolating a full length sequence using the two approaches described above.
  • Sequences are determined using the Sanger dideoxy method.
  • the encoded amino acid sequences may be deduced by routine methods.
  • antibodies may be raised against the polypeptide.
  • the antibodies are used to screen a ⁇ gt11 expression library made from cDNA copied from mRNA from cells in which the polypeptide may be expressed.
  • polypeptide encoded by the isolated nucleic acid may be desirable to express in order to determine that the polypeptide has the expected properties, for example expected ability to bind to a said phosphoinositide-binding polypeptide, for example TAPP.
  • a transgenic animal in which a said phosphoinositide-binding polypeptide gene is altered and/or a recombinant said phosphoinositide-binding polypeptide gene is present for example a rodent, in particular a mouse, may be useful in, for example, identifying polypeptides that interact with the said phosphoinositide-binding polypeptide.
  • the interacting polypeptide may be a receptor molecule, for example a receptor molecule present in/on the surface of a cell, for example a platelet, adipocyte, muscle or skin cell.
  • the receptor molecule may be a transmembrane polypeptide or complex, as know to those skilled in the art. It will be appreciated that known receptors, for example platelet integrin receptors, are not included.
  • screening assays which are capable of high throughput operation will be particularly preferred.
  • Examples may include cell based assays and protein-protein binding assays.
  • An SPA-based (Scintillation Proximity Assay; Amersham International) system may be used.
  • beads comprising scintillant and an interacting polypeptide which term it will be appreciated includes a polypeptide which capable of interacting with a polypeptide of the invention or fragment thereof and is a fragment of a polypeptide, for example a naturally occuring polypeptide, that is also capable of interacting with a polypeptide of the invention or fragment thereof) may be prepared.
  • the beads may be mixed with a sample comprising, for example, the phosphatidylinositol-binding polypeptide into which a radioactive label has been incorporated and with the test compound. Conveniently this is done in a 96-well format. The plate is then counted using a suitable scintillation counter, using known parameters for the particular radioactive label in an SPA assay. Only the radioactive label that is in proximity to the scintillant, ie only that bound to the phosphoinositide-binding polypeptide that is bound to the interacting polypeptide anchored on the beads, is detected.
  • Variants of such an assay for example in which the interacting polypeptide is immobilised on the scintillant beads via binding to an antibody or antibody fragment, may also be used.
  • Phosphoinositides or analogues thereof may be immobilised on SPA beads, for example using methods as described in Shirai et al (1998) Biochim Biophys Acta 1402(3), 292-302 or in Rao et al (1999) J Biol Chem 274, 37893-37900 .
  • screening assays of the invention are useful for identifying compounds which may be useful in the treatment of diabetes, defects of glycogen metabolism, cancer (including melanoma), inflammatory conditions, ischaemic conditions, for example stroke, thrombosis or tendency to thrombosis (for example useful as an antithrombotic agent).
  • the compound may be a drug-like compound or lead compound for the development of a drug-like compound for each of the above methods of identifying a compound. It will be appreciated that the said methods may be useful as screening assays in the development of pharmaceutical compounds or drugs, as well known to those skilled in the art.
  • drug-like compound is well known to those skilled in the art, and may include the meaning of a compound that has characteristics that may make it suitable for use in medicine, for example as the active ingredient in a medicament.
  • a drug-like compound may be a molecule that may be synthesised by the techniques of organic chemistry, less preferably by techniques of molecular biology or biochemistry, and is preferably a small molecule, which may be of less than 5000 daltons molecular weight.
  • a drug-like compound may additionally exhibit features of selective interaction with a particular protein or proteins and be bioavailable and/or able to penetrate cellular membranes, but it will be appreciated that these features are not essential.
  • lead compound is similarly well known to those skilled in the art, and may include the meaning that the compound, whilst not itself suitable for use as a drug (for example because it is only weakly potent against its intended target, non-selective in its action, unstable, difficult to synthesise or has poor bioavailability) may provide a starting-point for the design of other compounds that may have more desirable characteristics.
  • the compound may be a polypeptide that is capable of competing with the polypeptide of the invention for binding to the interacting polypeptide.
  • a screening method as described above may be useful in identifying polypeptides that may also interact with the interacting polypeptide, for example a receptor molecule.
  • reagents and conditions used in the method may be chosen such that the interactions between the said polypeptide and the interacting polypeptide are substantially the same as between the said polypeptide or a fragment therof and a naturally occuring interacting polypeptide in vivo .
  • the "drug-like compounds” and “lead compounds” identified in the screening assays of the invention are suitably screened in further screens to determine their potential usefulness in treating diabetes, defects of glycogen metabolism, cancer (including melanoma), inflammatory conditions, ischaemic conditions, for example stroke, or thrombosis or tendency to thrombosis. Additional screens which may be carried out include determining the effect of the compounds on blood glucose levels, tumour growth or blood clotting tendency/time, as appropriate. This may typically be done in rodents.
  • a further aspect of the invention is a kit of parts useful in carrying out a method, for example a detecting and/or quantifying method, of the invention.
  • a kit will comprise a said phosphoinositide-binding polypeptide of the preceding aspects of the invention (or a suitable fragment, thereof).
  • the phosphoinositide-binding polypeptide are as set out in relation to earlier aspects of the invention. It is particularly preferred that the phosphoinositide-binding polypeptide (or fragment thereof) is a naturally occurring polypeptide or naturally occurring allelic variant thereof.
  • the said phosphoinositide-binding polypeptide or fragment, used in the methods is one which is produced by recombinant DNA technology.
  • a method of the invention for example a compound screening method of the invention, in the presence of the phosphoinositide to which the said phosphoinositide-binding protein is capable of binding.
  • Expression of a constitutively active phosphoinositide (PI) kinase may be desirable in relation to a cell-based assay, in order to elevate the level of the appropriate phosphoinositide in the cell.
  • PI constitutively active phosphoinositide
  • (over)expression of a Class 1A PI3 kinase may be useful in relation to TAPP, as it may increase the level of PtdIns(3,4,5)P 3 and thereby the level of PtdIns(3,4)P 2 .
  • Overexpression of a Class II PI3 kinase may be useful in relation to PEPP or AtPH1, as it may increase the level of PtdIns3P, whilst overexpression of a PI4 kinase may be useful in relation to FAPP, as it may increase the level of PtdIns4P.
  • Overexpression of Fab1[38, 39] may be useful in, relation to centaurin- ⁇ 2, as it may increase the level of Ptd(3,5)P 2.
  • the said components in the method are those that have interactions or activities which are substantially the same as those of the said phosphoinositide-binding polypeptide or as the case may be but which may be more convenient to use in an assay.
  • fusions of the said phosphoinositide-binding polypeptide are particularly useful since said fusion may contain a moiety which may allow the fusion to be purified readily.
  • a further aspect of the invention provides a method of detecting and/or quantifying PtdIns(3,4)P 2 , in a sample wherein the sample is exposed to a polypeptide capable of binding to PtdIns(3,4)P2, but not capable of binding to PtdIns(3,4,5)P 3 according to the preceding aspects of the invention and the binding of the said polypeptide to any PtdIns(3,4)P 2 present is detected.
  • Preferences for the said polypeptide are as indicated in relation to the preceding and following aspects of the invention. Methods of detecting binding of the PtdIns(3,4)P 2 to the said polypeptide are discussed above and in Examples 1 and 3.
  • the polypeptides may be used to determine the location of the PtdIns(3,4)P 2 using in situ techniques, as well known to those skilled in the art.
  • the cells may be living cells, or fixed using conventional methods, for example formaldehyde fixing.
  • the said polypeptide comprises a chromophore, for example a green fluorescent protein moiety (GFP; including mutated GFPs, for example blue, yellow or cyan fluorescent proteins), for example as a fusion protein which is expressed in the cell, as well known to those skilled in the art.
  • GFP green fluorescent protein moiety
  • GFPs are produced naturally by Aequorea victoria but, as is well known in the art and described, for example, in Mitra et al (1996) Gene 173, 13-17 ; Cubitt et al (1995) Trends Biochem. Sci. 20, 448-454 ; Miyawaki et al (1997) Nature 388, 882-887 ; Patterson et al (1997) Biophys J. 73, 2782-2690 ; Heim & Tsien (1996) Curr. Biol. 6, 178-182 ; and Heim et al (1995) Nature 373, 663-664 , mutant GFPs are available which have modified spectral characteristics. Certain GFPs and mutant GFPs are available from Clontech Laboratories UK Ltd, Wade Road, Basingstoke, Hants RG24 8NE.
  • the methods may be used in assays for detecting or quantifying (measuring) enzyme activity, for example lipid phosphatases or inositol lipid kinases, for example Fablp (a stress-activated phosphatidylinositol 3-phosphate 5-kinase), which converts PtdIns3P to PtdIns(3,5)P 2.
  • a PH domain which binds to PtdIns3P (for example the PH domain of PEPP1 or AtPH1) may be used to monitor the level of PtdIns3P and thereby Fablp activity. This is discussed further in Example 3.
  • Such a lipid kinase/phosphatase assay may be performed in vitro (for example using techniques described above and in Examples 1 and 3) or in vivo, for example in cells, using techniques as described above.
  • the methods may be used in identifying modulators (for example inhibitors or activators) of the enzyme activity, as will be apparent to those skilled in the art.
  • the invention provides a method for identifying a modulator of a lipid kinase or phosphatase activity wherein the lipid kinase or phosphatase activity is measured in the presence (and preferably also in the absence, or in the presence of more than one concentration) of the compound using such a method.
  • the invention further provides a kit of parts useful in carrying out such a detection/quantification or screening method.
  • Suitable components for such a kit include reagents and enzymes of the types mentioned in Example 3, for example a PH domain of the invention and a phosphoinositide which binds to the said PH domain or a lipid which is converted into a phosphoinositide which binds to the said PH domain by an enzyme, for example lipid kinase or phosphatase.
  • a further aspect of the invention provides a composition comprising a polypeptide capable of binding specifically to PtdIns(3,4)P 2, but not capable of binding to PtdIns(3,4,5)P 3 , wherein the polypeptide comprises a PH domain capable of binding to PfdIns(3,4)P 2 but not capable of binding to PtdIns(3,4,5)P 3 wherein the polypeptide comprises the amino acid sequence or or a fragment thereof capable of binding specifically to PtdIns(3,4)P 2 but not capable of binding to PtdIns(3,4,5)P 3 or the polypeptide may comprise a fusion of a said fragment thereof, wherein the composition comprises a protein content that consists of at least 50% by weight of said polypeptide.
  • a PI 4-kinase polypeptide (or recombinant polypeptide comprising a PH domain therefrom) as described in Stevenson et al (1998) J Biol Chem 273, 22761-22767 is capable of binding to PtdIns(3,4)P 2 , PtdIns3P, PtdIns4P and/or PtdIns(3,5)P 2 )but is not capable of binding to PtdIns(3,4,5)P 3.
  • polypeptides described in Stevenson et al (1998) (ie PI 4 kinases and PH domains thereof from Arabidopsis, carrot, yeast STT4, rat, human PIK4K ⁇ and bovine brain PI4K200 are excluded from the polypeptides of the invention These polypeptides are further not considered to comprise a PH domain which has at least five of the six residues of a P utative P tdIns(3,4,5)P 3 Binding M otif (PPBM).
  • PPBM P utative P tdIns(3,4,5)P 3 Binding M otif
  • PLC ⁇ 1 is capable of binding to PtdIns(3,4)P 2 , PtdIns3P, PtdIns4P and/or PtdIns(3,5)P 2 ) but is not capable of binding to PtdIns(3,4,5)P 3 .
  • PLC ⁇ 1 is excluded from the polypeptides of the invention.
  • polypeptide of the invention may be useful in accordance with the uses or screens of the preceding aspects of the invention, as indicated above.
  • polypeptides of the invention include TAPP2 fragments thereof, or fusions of fragments, for example a fragment comprising a phosphoinositide-binding PH domain. The said fragment, or fusion retains the phosphoinositide binding properties of the polypeptide of the invention from which it is derived/derivable, as discussed further below.
  • composition comprising a polypeptide comprising the amino acid sequence (partial human TAPP2 amino acid sequence) or (mouse TAPP2 amino acid sequence; see also Accession No AF286161) or a fragment, thereof, or a fusion of a said fragment, thereof.
  • TAPP polypeptides include the chicken TAPP2 sequence as given in Accession No AF302149.
  • Human TAPP2 may have the sequence given in Accession No AF 286164, which is a fragment of the sequence given above, as follows:
  • Standard IUPAC one and three letter codes are used for amino acid sequences used in the specification, and the amino acid sequences are listed N-terminal to C-terminal as is conventional.
  • substantially pure we mean that the said polypeptide is substantially free of other proteins.
  • any composition that includes at least 30% of the protein content by weight as the said polypeptide, preferably at least 50%, more preferably at least 70%, still more preferably at least 90% and most preferably at least 95% of the protein content is the said polypeptide.
  • the invention also includes compositions comprising the said polypeptide and a contaminant wherein the contaminant comprises less than 70% of the composition by weight, preferably less than 50% of the composition, more preferably less than 30% of the composition, still more preferably less than 10% of the composition and most preferably less than 5% of the composition by weight.
  • the invention also includes the substantially pure said polypeptide when combined with other components ex vivo, said other components not being all of the components found in the cell in which said polypeptide is found.
  • the polypeptides of the invention can be produced using recombinant DNA technology.
  • Variants may be made using the methods of protein engineering and site-directed mutagenesis well known in the art using the recombinant polynucleotides described below.
  • fragment of said polypeptide we include any fragment which retains activity or which is useful in some other way, for example, for use in raising antibodies or in a binding or other assay, or which fragment may have other functions as described in more detail below. Preferred fragments of TAPP are discussed further below.
  • fusion of said polypeptide we include said polypeptide fused to any other polypeptide.
  • the said polypeptide may be fused to a polypeptide such as glutathione-S-transferase (GST) or protein A in order to facilitate purification of said polypeptide. Examples of such fusions are well known to those skilled in the art.
  • the said polypeptide may be fused to an oligo-histidine tag such as His6 or to an epitope recognised by an antibody such as the well known Myc tag epitope. Fusions to any fragment of said polypeptide are also included in the scope of the invention.
  • fusions or fragments thereof which retain desirable properties, such as binding properties (for example, the ability to bind to a particular phosphoinositide) or the ability to change subcellular location in response to stress, insulin or growth factor signalling (in an intact cell) or other biological functions, of the said polypeptide (for example TAPP) are particularly preferred. It is also particularly preferred if the fusions are one which are suitable for use in the screening assays described earlier.
  • fusions which retain desirable properties, such as binding properties or other biological functions, of the said polypeptide are particularly preferred. It is also particularly preferred if the fusions are one which are suitable for use in the screening assays described above. It will be appreciated that before the present, invention, no requirement for producing any of the said polypeptides, or for variants or fusions or derivatives thereof, had not been appreciated in the art since their involvement in phosphoinositide signalling was not known. In particular it was not appreciated that the said polypeptides and variants and fusions thereof would be useful in screening methods for drugs and drug-like compounds.
  • variants of the polypeptide we include insertions, deletions and substitutions, either conservative or non-conservative. In particular we include variants of the polypeptide where such changes do not substantially alter the activity of the said polypeptide. In particular we include variants of the polypeptide where such changes do not substantially alter the activity, for example the binding activity (for example to a phosphoinositide) of the said polypeptide. Variants of the said polypeptides do not include polypeptides which have the amino acid sequence of known polypeptides comprising a PH domain.
  • substantially all of the sequence shown above for example substantially full-length TAPP, PEPP or FAPP
  • substantially full-length is meant at least 80%, preferably 90%, still more preferably 95%, 98% or 100% (ie all) of the said sequence.
  • substantially full-length is meant comprising at least 80%, preferably 90%, still more preferably 95%, 98% or 100% (ie all) of the sequence of the full length polypeptide.
  • substitutions is intended combinations such as Gly, Ala; Val, Ile, Leu; Asp, Glu; Asn, GIn; Ser, Thr; Lys, Arg; and Phe, Tyr.
  • polypeptide variant has an amino acid sequence which has at least 65% identity with either amino acid sequence given above, more preferably at least 75%, still more preferably at least 90%, yet more preferably at least 95%, and most preferably at least 98% or 99% identity with the appropriate amino acid sequence given above, most preferably with the amino acid sequence given above for human TAPP.
  • the polypeptide variant has an amino acid sequence which has at least 90% identity with the amino acid sequence given above, more preferably at least 92%, still more preferably at least 95%, yet more preferably at least 96%, and most preferably at least 98% or 99% identity with the amino acid sequence given above.
  • the percent sequence identity between two polypeptides may be determined using suitable computer programs, for example the GAP program of the University of Wisconsin Genetic Computing Group and it will be appreciated that percent identity is calculated in relation to polypeptides whose sequences have been aligned optimally.
  • the alignment may alternatively be carried out using the Clustal W program ( Thompson, J.D., Higgins, D.G. and Gibson, T.J. (1994), Clustal-W - improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position specific gap penalties and weight matrix choice. Nuc. Acid Res. 22, 4673-4680 ).
  • the parameters used may be as follows:
  • “Fragments” and “variants” also include those which are useful to prepare antibodies which will specifically bind the said polypeptide or mutant forms thereof lacking the function of the native polypeptide.
  • Such variants and fragments will usually include at least one region of at least five consecutive amino acids which has at least 90% homology with the most homologous five or more consecutive amino acids region of the said polypeptide (ie when comparing forms of the polypeptide from different species).
  • a fragment is less than 100% of the whole polypeptide.
  • TAPP1 particularly human TAPP1 immunogens: YVDRQNRICGFLDIEENENSGK (this one would also be expected to recognise TAPP2) and RYTSRAGECSTYVGSHANVPS.
  • TAPP2 particularly mouse TAPP2
  • immunogens RVRHRSEPQHPKEKPFVFNL and KRGLCKAPSVASSWQPWTPVKK.
  • TAPP 1 and TAPP2 are most dissimilar in the C-terminal region (excluding the extreme C-terminus), as is apparent from Figure 3A . Accordingly, a peptide with a sequence derived from the less-conserved C-terminal region of TAPP1 or TAPP2 may be useful in preparing antibodies that are specific for TAPP1 or TAPP2, respectively. A peptide with a sequence derived from the more conserved N-terminal region of TAPP1/TAPP2 may be useful in preparing antibodies that react with both TAPP1 and TAPP2.
  • polypeptide of the invention may be modified by known polypeptide modification techniques. These include the techniques disclosed in US Patent No 4,302,386 issued 24 November 1981 to Stevens . Such modifications may enhance the immunogenicity of the antigen, or they may have no effect on such immunogenicity. For example, a few amino acid residues may be changed.
  • the antigen of the invention may contain one or more amino acid sequences that are not necessary to its immunogenicity. Unwanted sequences can be removed by techniques well known in the art. For example, the sequences can be removed via limited proteolytic digestion using enzymes such as trypsin or papain or related proteolytic enzymes.
  • polypeptides corresponding to antigenic parts of the polypeptide may be chemically synthesised by methods well known in the art. These include the methods disclosed in US Patent No 4,290,944 issued 22 September 1981 to Goldberg .
  • polypeptide of the invention includes a class of modified polypeptides, including synthetically derived polypeptides or fragments of the original polypeptide, having common elements of origin, structure, and immunogenicity that are within the scope of the present invention.
  • An additional embodiment of this aspect of the invention relates to a peptide or polypeptide which has the amino acid sequence of an epitope-bearing portion of a polypeptide of the invention, ie having an amino acid sequence described above.
  • Such peptides or polypeptides include portions of a polypeptide of the invention with at least six or seven, preferably at least nine, and more preferably at least about 30 amino acids to about 50 amino acids, although epitope-bearing polypeptides of any length up to and including the complete amino acid sequence of a polypeptide of the invention described above also are included in the invention.
  • a particular embodiment of the invention provides a substantially pure TAPP polypeptide which consists of the amino acid sequence indicated above for human or mouse TAPP2 or naturally occurring allelic variants thereof
  • a preferred fragment of the TAPP polypeptide of the invention comprises the amino acid sequence of amino acids 1 to 147 of any of the given TAPP amino acid sequences.
  • This fragment comprises the N-teiminal PH domain of TAPP. It is further preferred that the fragment does not comprise the amino acid sequence of about amino acids 190 to about 290 of the given amino acid sequence of TAPP.
  • This fragment comprises the N-terminal PH domain of TAPP1 and does not comprise the C-terminal PH domain of TAPP1.
  • a further preferred fragment of the polypeptide of the invention comprises the amino acid sequence of amino acids 95 to 404 of any of the given TAPP amino acid sequences.
  • This fragment comprises the C-terminal PH domain of TAPP1. It is further preferred that the fragment does not comprise the amino acid sequence of about amino acids 10 to 111 of the given amino acid sequence.
  • This fragment comprises the C-terminal PH domain of TAPP1 and does not comprise the entire N-terminal domain of TAPP1.
  • Preferred fusions of these fragments include fusions as described in Example 1, for example fusions in which the said fragment has an N-terminal GST tag followed by a myc epitope tag or a FLAG (DYKDDDDK) epitope tag fused to the N-terminus of the said fragment.
  • a variant of the TAPP polypeptide of the invention which may be useful is a variant (or fragment, derivative or fusion of such a variant) wherein the residue equivalent to Arg212 of the given human TAPP1 amino acid sequence is mutated, for example to a leucine residue.
  • Such a variant may be less able or unable to bind to PtdIns(3,4)P 2 (or other phosphoinositide), as described in Example 1.
  • variants of the polypeptide of the invention which may be useful are variants (or fragments, derivatives or fusions of such a variant) wherein the residue equivalent to any of the lysine or arginine residues of the PPBP is mutated to an acidic residue, for example glutamate or to a large hydrophobic residue, for example methionine.
  • an acidic residue for example glutamate or to a large hydrophobic residue, for example methionine.
  • Such a variant may be less able or unable to bind to a phosphoinositide, as described in Example 1.
  • fragments and variants may be useful in screening assays, medicine and/or in investigating the involvement of TAPP or other polypeptide of the invention in normal and diseased cells.
  • a fragment of TAPP comprising the N-terminal (putative protein-binding) PH domain but not the C-terminal (phosphoinositide-binding) PH domain or a fragment of TAPP comprising the N-terminal PH domain but not the C-terminal PH domain may be capable of acting as an inhibitor, for example a dominant-negative inhibitor, of signalling via a signalling pathway in which TAPP may be involved, as discussed further below, for example signalling via an integrin receptor or a growth factor receptor.
  • a variant of TAPP in which any of the conserved Lys/Arg sites in the PPBM is replaced with an acidic or hydrophobic residue, for example leucine, may act as a dominant negative mutant, which may bind to interacting polypeptides (for example via the N-terminal PH domain) but not to the phosphoinositide.
  • a fragment may be useful, for example, as an anti-cancer agent or in the promotion of apoptosis. Promotion of apoptosis may be beneficial in the resolution of inflammation.
  • Inhibition of TAPP activity may inhibit platelet activation, which may be useful in reducing or preventing thrombosis. This may be important in patients at risk of thrombosis (for example obese patients or those with a history of thrombosis) and/or before, during or after surgery.
  • Over-expression of a substantially full-length native said polypeptide may be useful in increasing signalling in which the said polypeptide is involved and therefore may also be useful in the treatment of diabetes or defects of glycogen regulation. It may also be useful in reducing apoptosis; thus, it may be useful in treating a patient in need of protection against apoptosis. Reducing apoptosis may be useful following ischaemic injury, for example stroke or myocardial infarction, and in tissue repair. It may also be useful in the treatment of patient before, after or during heart surgery.
  • a fusion of a polypeptide, variant or fragment of the invention wherein the fusion comprises a GST and/or FLAG or myc epitope portion may be particularly useful.
  • a GST tag may be useful in purifying or detecting the fusion protein, as described in Example 1, for example in detecting the interaction between the fusion protein and a phospholipid.
  • the fragment of the said polypeptide, or the fusion of the fragment has at least 30% of the PtdIns(3,4)P 2 , binding affinity of the said polypeptide, for example TAPP but is not capable of binding to PtdIns(3,4,5)P 3 . It is more preferred if the fragment of the said polypeptide, or the fusion of the fragment has at least 50%, preferably at least 70% and more preferably at least 90% of the phosphoinositide binding activity of the said polypeptide, for example TAPP.
  • fusions or fragments which are devoid of one or more binding activities as set out above may nevertheless be useful, for example as described above or by interacting with another polypeptide, or as antigens in raising antibodies.
  • Methods of measuring the binding affinity with phosphoinositides are described, for example, in Example 1 below. Methods of measuring protein-protein interactions are well known to those skilled in the art and are discussed above.
  • residue equivalent to a particular residue, for example the residue equivalent to Arg212 of human TAPP1, is included the meaning that the amino acid residue occupies a position in the native two or three dimensional structure of a polypeptide corresponding to the position occupied by the said particular residue, for example Arg212, in the native two or three dimensional structure of full-length human TAPP1.
  • the residue equivalent to a particular residue may be identified by alignment of the sequence of the polypeptide with that of full-length human TAPP1 in such a way as to maximise the match between the sequences.
  • the alignment may be carried out by visual inspection and/or by the use of suitable computer programs, for example the GAP program of the University of Wisconsin Genetic Computing Group, which will also allow the percent identity of the polypeptides to be calculated, or using the Align program ( Pearson (1994) in: Methods in Molecular Biology, Computer Analysis of Sequence Data, Part II (Griffin, AM and Griffin, HG eds) pp 365-389, Humana Press, Clift on).
  • residues identified in this manner are also "equivalent residues”.
  • Peptides may be synthesised by the Fmoc-polyamide mode of solid-phase peptide synthesis as disclosed by Lu et al (1981) J. Org. Chem. 46, 3433 and references therein. Temporary N-amino group protection is afforded by the 9-fluorenylmethyloxycarbonyl (Fmoc) group. Repetitive cleavage of this highly base-labile protecting group is effected using 20% piperidine in N,N-dimethylformamide.
  • Side-chain functionalities may be protected as their butyl ethers (in the case of serine threonine and tyrosine), butyl esters (in the case of glutamic acid and aspartic acid), butyloxycarbonyl derivative (in the case of lysine and histidine), trityl derivative (in the case of cysteine) and 4-methoxy-2,3,6-trimethylbenzenesulphonyl derivative (in the case of arginine).
  • glutamine or asparagine are C-terminal residues, use is made of the 4,4'-dimethoxybenzhydryl group for protection of the side chain amido functionalities.
  • the solid-phase support is based on a polydimethylacrylamide polymer constituted from the three monomers dimethylacrylamide (backbone-monomer), bisacryloylethylene diamine (cross linker) and acryloylsarcosine methyl ester (functionalising agent).
  • the peptide-to-resin cleavable linked agent used is the acid-labile 4-hydroxymethyl-phenoxyacetic acid derivative. All amino acid derivatives are added as their preformed symmetrical anhydride derivatives with the exception of asparagine and glutamine, which are added using a reversed N,N-dicyclohexylcarbodiimide/1-hydroxybenzotriazole mediated coupling procedure.
  • Any scavengers present are removed by a simple extraction procedure which on lyophilisation of the aqueous phase affords the crude peptide free of scavengers.
  • Reagents far peptide synthesis are generally available from Calbiochem-Novabiochem (UK) Ltd, Nottingham NG7 2QJ, UK.
  • Purification may be effected by any one, or a combination of, techniques such as size exclusion chromatography, ionexchange chromatography and (principally) reverse-phase high performance liquid chromatography.
  • Analysis of peptides may be carried out using thin layer chromatography, reverse-phase high performance liquid chromatography, amino-acid analysis after acid hydrolysis and by fast atom bombardment (FAB) mass spectrometric analysis.
  • FAB fast atom bombardment
  • a further embodiment of the invention provides a recombinant polynucleotide suitable for expressing a said phosphoinositide-binding protein of the preceding aspects of invention or suitable for expressing a fragment of said polypeptide or a fusion of a said fragment.
  • the polynucleotide is a polynucleotide that may be translated to form the polypeptide, for example RNA, or that the polynucleotide (which is preferably DNA) encoding the polypeptide of the invention is inserted into an expression vector, such as a plasmid, in proper orientation and correct reading frame for expression.
  • an expression vector such as a plasmid
  • the polynucleotide may be linked to the appropriate transcriptional and translational regulatory control nucleotide sequences recognised by any desired host; such controls may be incorporated in the expression vector.
  • a further embodiment of the invention is a replicable vector suitable for expressing a polypeptide as defined in the preceding aspect of the invention.
  • the replicable vector may be suitable for expressing a fusion of the said phosphoinositide-binding polypeptide, in particular a GST fusion.
  • vectors suitable for replication in mammalian/eukaryotic cells are well known to those skilled in the art. It will be appreciated that a vector may be suitable for replication in both prokaryotic and eukaryotic cells.
  • polynucleotide comprises the nucleotide sequence: (partial human TAPP2) or (mouse TAPP2) or a fragment as fusion thereof.
  • Example 1 References for full length sequences of centaurin- ⁇ 2 and AtPH1 are given in Example 1, for example in Table 1. Polynucleotides encoding full-length centaurin- ⁇ 2 or AtPH1 are excluded from the polynucleotides of the invention.
  • sequences encoding other full length TAPP polypeptides may be obtained by routine use of methods well known to those skilled in the art, making use of the sequences shown above.
  • PCR methods may be used, particularly methods developed to generate 5' cDNA sequences (for example, the "RACE” method, as well known to those skilled in the art).
  • RACE 5' cDNA sequences
  • sequence database analysis for example EST database analysis and sequencing, as well known to those skilled in the art.
  • an expressed sequence tag (EST) clone is not a recombinant polynucleotide as defined above as it lacks sequences necessary for the translation and therefore expression of the expressed sequence tag.
  • EST sequences may be cloned in the vector Uni-ZAP XR, pT7T3D-Pac, pBluescript SK-, Lafmid BA or pCMV-SPORT2 vector.
  • a polynucleotide comprising a fragment of the recombinant polynucleotide encoding a polypeptide of the invention or a variant, fragment, fusion or derivative may also be useful.
  • the polynucleotide comprises a fragment which is at least 10 nucleotides in length, more preferably at least 14 nucleotides in length and still more preferably at least 18 nucleotides in length.
  • Such polynucleotides are useful as PCR primers.
  • a polynucleotide complementary to the polynucleotide (or a fragment thereof) encoding a polypeptide of the invention or a variant, fragment, fusion or derivative may also be useful. Such complementary polynucleotides are well known to those skilled in the art as antisense polynucleotides.
  • the polynucleotide or recombinant polynucleotide of the invention may be DNA or RNA, preferably DNA.
  • the polynucleotide may or may not contain introns in the coding sequence; preferably the polynucleotide is a cDNA.
  • a "variation" of the polynucleotide includes one which is (i) usable to produce a protein or a fragment thereof which is in turn usable, for example a processed polypeptide as described above, or to prepare antibodies which specifically bind to the protein encoded by the said polynucleotide or (ii) an antisense sequence corresponding to the gene or to a variation of type (i) as just defined.
  • different codons can be substituted which code for the same amino acid(s) as the original codons.
  • the substitute codons may code for a different amino acid that will not affect the activity or immunogenicity of the protein or which may improve or otherwise modulate its activity or immunogenicity.
  • site-directed mutagenesis or other techniques can be employed to create single or multiple mutations, such as replacements, insertions, deletions, and transpositions, as described in Botstein and Shortle, "Strategies and Applications of In Vitro Mutagenesis" Science, 229: 193-210 (1985 ). Since such modified polynucleotides can be obtained by the application of known techniques to the teachings contained herein, such modified polynucleotides are within the scope of the claimed invention.
  • polynucleotide sequence (or fragments thereof) encoding a polypeptide of the invention can be used to obtain other polynucleotide sequences that hybridise with it under conditions of high stringency.
  • Such polynucleotides includes any genomic DNA.
  • the polynucleotide of the invention includes polynucleotide that shows at least 80%, preferably 85%, and more preferably at least 90% and most preferably at least 95% homology with the polynucleotide identified in the method of the invention, provided that such homologous polynucleotide encodes a polypeptide which is usable in at least some of the methods described below or is otherwise useful.
  • polynucleotide sequence (or fragments thereof) encoding a polypeptide of the invention can be used to obtain other polynucleotide sequences that hybridise with it under conditions of high stringency.
  • Such polynucleotides includes any genomic DNA.
  • the polynucleotide of the invention includes polynucleotide that shows at least 60%, preferably 70%, and more preferably at least 80% and most preferably at least 90% homology with the polynucleotide identified in the method of the invention, provided that such homologous polynucleotide encodes a polypeptide which is usable in at least some of the methods described below or is otherwise useful.
  • a polynucleotide encoding full length centaurin- ⁇ 2 or AtPH1 is not a polynucleotide of the invention.
  • Per cent homology can be determined by, for example, the GAP program of the University of Wisconsin Genetic Computer Group.
  • DNA-DNA, DNA-RNA and RNA-RNA hybridisation may be performed in aqueous solution containing between 0.11XSSC and 6XSSC and at temperatures of between 55°C and 70°C. It is well known in the art that the higher the temperature or the lower the SSC concentration the more stringent the hybridisation conditions. By “high stringency” we mean 2XSSC and 65°C. 1XSSC is 0.15M NaCl/0.015M sodium citrate. Polynucleotides which hybridise at high stringency are included within the scope of the claimed invention.
  • “Variations" of the polynucleotide also include polynucleotide in which relatively short stretches (for example 20 to 50 nucleotides) have a high degree of homology (at least 80% and preferably at least 90 or 95%) with equivalent stretches of the polynucleotide of the invention even though the overall homology between the two polynucleotides may be much less. This is because important active or binding sites may be shared even when the general architecture of the protein is different.
  • a desirable way to modify the DNA encoding a polypeptide of the invention is to use the polymerase chain reaction as disclosed by Saiki et al (1988) Science 239, 487-491 .
  • This method may be used for introducing the DNA into a suitable vector, for example by engineering in suitable restriction sites, or it may be used to modify the DNA in other useful ways as is known in the art.
  • the DNA to be enzymatically amplified is flanked by two specific primers which themselves become incorporated into the amplified DNA.
  • the said specific primers may contain restriction endonuclease recognition sites which can be used for cloning into expression vectors using methods known in the art.
  • the DNA (or in the case of retroviral vectors, RNA) is then expressed in a suitable host to produce a polypeptide comprising the compound of the invention.
  • the DNA encoding the polypeptide constituting the compound of the invention may be used in accordance with known techniques, appropriately modified in view of the teachings contained herein, to construct an expression vector, which is then used to transform an appropriate host cell for the expression and production of the polypeptide of the invention.
  • Such techniques include those disclosed in US Patent Nos.
  • DNA (or in the case of retroviral vectors, RNA) encoding the polypeptide constituting the compound of the invention may be joined to a wide variety of other DNA sequences for introduction into an appropriate host.
  • the companion DNA will depend upon the nature of the host, the manner of the introduction of the DNA into the host, and whether episomal maintenance or integration is desired.
  • the DNA is inserted into an expression vector, such as a plasmid, in proper orientation and correct reading frame for expression.
  • an expression vector such as a plasmid
  • the DNA may be linked to the appropriate transcriptional and translational regulatory control nucleotide sequences recognised by the desired host, although such controls are generally available in the expression vector.
  • the vector is then introduced into the host through standard techniques. Generally, not all of the hosts will be transformed by the vector. Therefore, it will be necessary to select for transformed host cells.
  • One selection technique involves incorporating into the expression vector a DNA sequence, with any necessary control elements, that codes for a selectable trait in the transformed cell, such as antibiotic resistance.
  • the gene for such selectable trait can be on another vector, which is used to co-transform the desired host cell.
  • Host cells that have been transformed by the recombinant DNA of the invention are then cultured for a sufficient time and under appropriate conditions known to those skilled in the art in view of the teachings disclosed herein to permit the expression of the polypeptide, which can then be recovered.
  • bacteria for example E . coli and Bacillus subtilis
  • yeasts for example Saccharomyces cerevisiae
  • filamentous fungi for example Aspergillus
  • plant cells animal cells and insect cells.
  • the vectors include a prokaryotic replicon, such as the ColEl ori, for propagation in a prokaryote, even if the vector is to be used for expression in other, non-prokaryotic, cell types.
  • the vectors can also include an appropriate promoter such as a prokaryotic promoter capable of directing the expression (transcription and translation) of the genes in a bacterial host cell, such as E . coli, transformed therewith.
  • a promoter is an expression control element formed by a DNA sequence that permits binding of RNA polymerase and transcription to occur.
  • Promoter sequences compatible with exemplary bacterial hosts are typically provided in plasmid vectors containing convenient restriction sites for insertion of a DNA segment of the present invention.
  • Typical prokaryotic vector plasmids are pUC18, pUC19, pBR322 and pBR329 available from Biorad Laboratories, (Richmond, CA, USA) and pTrc99A and pKK223-3 available from Pharmacia, Piscataway, NJ, USA.
  • a typical mammalian cell vector plasmid is pSVL available from Pharmacia, Piscataway, NJ, USA. This vector uses the SV40 late promoter to drive expression of cloned genes, the highest level of expression being found in T antigen-producing cells, such as COS-1 cells.
  • an inducible mammalian expression vector is pMSG, also available from Pharmacia. This vector uses the glucocorticoid-inducible promoter of the mouse mammary tumour virus long terminal repeat to drive expression of the cloned gene.
  • the pEBG-2T expression vector may be used to express GST fusion proteins in eukaryotic cells, for example in 293 cells (human embryonic kidney cells).
  • Useful yeast plasmid vectors are pRS403-406 and pRS413-416 and are generally available from Stratagene Cloning Systems, La Jolla, CA 92037, USA.
  • Plasmids pRS403, pRS404, pRS405 and pRS406 are Yeast Integrating plasmids (YIps) and incorporate the yeast selectable markers HIS3, TRP1, LEU2 and URA3.
  • Plasmids pRS413-416 are Yeast Centromere plasmids (YCps).
  • Useful yeast plasmid vectors are pRS403-406 and pRS413-416 and are generally available from Stratagene Cloning Systems, La Jolla, CA 92037, USA.
  • Plasmids pRS403, pRS404, pRS405 and pRS406 are Yeast Integrating plasmids (YIps) and incorporate the yeast selectable markers HIS3, TRP1, LEU2 and URA3.
  • Plasmids pRS413-416 are Yeast Centromere plasmids (YCps).
  • the present invention also relates to a host cell transformed with a polynucleotide vector construct of the present invention.
  • the host cell can be either prokaryotic or eukaryotic.
  • Bacterial cells are preferred prokaryotic host cells and typically are a strain of E. coli such as, for example, the E . coli strains DH5 available from Bethesda Research Laboratories Inc., Bethesda, MD, USA, and RR1 available from the American Type Culture Collection (ATCC) of Rockville, MD, USA (No ATCC 31343).
  • Preferred eukaryotic host cells include yeast, insect and mammalian cells, preferably vertebrate cells such as those from a mouse, rat, monkey or human fibroblastic cell line.
  • Yeast host cells include YPH499, YPH500 and YPH501 which are generally available from Stratagene Cloning Systems, La Jolla, CA 92037, USA.
  • Preferred mammalian host cells include Chinese hamster ovary (CHO) cells available from the ATCC as CCL61, NIH Swiss mouse embryo cells NIH/3T3 available from the ATCC as CRL 1658, and monkey kidney-derived COS-1 cells available from the ATCC as CRL 1650.
  • Preferred insect cells are Sf9 cells which can be transfected with baculovirus expression vectors.
  • Transformation of appropriate cell hosts with a DNA construct of the present invention is accomplished by well known methods that typically depend on the type of vector used. With regard to transformation of prokaryotic host cells, see, for example, Cohen et al(1972) Proc. Natl. Acad. Sci. USA 69, 2110 and Sambrook et al(1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY . Transformation of yeast cells is described in Sherman et al(1986) Methods In Yeast Genetics, A Laboratory Manual, Cold Spring Harbor, NY . The method of Beggs (1978) Nature 275, 104-109 is also useful.
  • reagents useful in transfecting such cells for example calcium phosphate and DEAE-dextran or liposome formulations, are available from Stratagene Cloning Systems, or Life Technologies Inc., Gaithersburg, MD 20877, USA.
  • Electroporation is also useful for transforming and/or transfecting cells and is well known in the art for transforming yeast cell, bacterial cells, insect cells and vertebrate cells.
  • bacterial species may be transformed by the methods described in Luchansky et al(1988) Mol. Microbiol. 2, 637-646 incorporated herein by reference. The greatest number of transformants is consistently recovered following electroporation of the DNA-cell mixture suspended in 2.5X PEB using 6250V per cm at 25:FD.
  • Successfully transformed cells ie cells that contain a DNA construct of the present invention
  • cells resulting from the introduction of an expression construct of the present invention can be grown to produce the polypeptide of the invention.
  • Cells can be harvested and lysed and their DNA content examined for the presence of the DNA using a method such as that described by Southern (1975) J. Mol. Biol. 98, 503 or Berent et al(1985) Biotech. 3, 208 .
  • the presence of the protein in the supernatant can be detected using antibodies as described below.
  • successful transformation can be confirmed by well known immunological methods when the recombinant DNA is capable of directing the expression of the protein.
  • cells successfully transformed with an expression vector produce proteins displaying appropriate antigenicity. Samples of cells suspected of being transformed are harvested and assayed for the protein using suitable antibodies.
  • the present invention also contemplates a culture of those cells, preferably a monoclonal (clonally homogeneous) culture, or a culture derived from a monoclonal culture, in a nutrient medium.
  • a further aspect of the invention provides a method of making the polypeptide of the preceding aspect of the invention the method comprising culturing a host cell comprising a recombinant polynucleotide or a replicable vector which encodes said polypeptide, and isolating said polypeptide from said host cell.
  • Methods of cultivating host cells and isolating recombinant proteins are well known in the art.
  • the invention also includes a polypeptide, or a variant, fragment, derivative or fusion thereof, or fusion of a said variant or fragment or derivative obtainable by the above method of the invention.
  • a still further aspect of the invention provides an antibody reactive towards a polypeptide of the preceding aspect of the invention, for example TAPP or a fragment thereof. It is preferred that the antibody is not an antibody reactive towards centaurin- ⁇ 2 or AtPH1.
  • the antibody does not react substantially with another polypeptide comprising a PH domain. Accordingly, it may be preferred if peptides based on the TAPP sequence are used which vary significantly from any peptides found in any other PH domains, for example in the polypeptides indicated in part A of Table 1.
  • Antibodies reactive towards the said polypeptide of the invention may be made by methods well known in the all In particular, the antibodies may be polyclonal or monoclonal.
  • Suitable monoclonal antibodies which are reactive towards the said polypeptide may be prepared by known techniques, for example those disclosed in “ Monoclonal Antibodies: A manual of techniques", H Zola (CRC Press, 1988 ) and in “ Monoclonal Hybridoma Antibodies: Techniques and Applications", SGR Hurrell (CRC Press, 1982 ).
  • the antibody is raised using any suitable peptide sequence obtainable from the given amino acid sequence, for example of TAPP. It is preferred if polyclonal antipeptide antibodies are made.
  • an antibody of the invention is capable of preventing or disrupting the interaction between a polypeptide of the invention or a fragment thereof and an interacting polypeptide identified by the method of the invention describe above, or a phosphoinositide. Such antibodies are believed to be useful in medicine, for example in treating cancer or promoting apoptosis.
  • Peptides in which one or more of the amino acid residues are chemically modified, before or after the peptide is synthesised may be used providing that the function of the peptide, namely the production of specific antibodies in vivo, remains substantially unchanged.
  • modifications include forming salts with acids or bases, especially physiologically acceptable organic or inorganic acids and bases, forming an ester or amide of a terminal carboxyl group, and attaching amino acid protecting groups such as N-t-butoxycarbonyl. Such modifications may protect the peptide from in vivo metabolism.
  • the peptides may be present as single copies or as multiples, for example tandem repeats. Such tandem or multiple repeats may be sufficiently antigenic themselves to obviate the use of a carrier.
  • the peptide may be formed as a loop, with the N-terminal and C-terminal ends joined together, or to add one or more Cys residues to an end to increase antigenicity and/or to allow disulphide bonds to be formed.
  • a carrier preferably a polypeptide
  • the arrangement is preferably such that the peptide of the invention forms a loop.
  • a carrier function should be present in any immunogenic formulation in order to stimulate, or enhance stimulation of, the immune system. It is thought that the best carriers embody (or, together with the antigen, create) a T-cell epitope.
  • the peptides may be associated, for example by cross-linking, with a separate carrier, such as serum albumins, myoglobins, bacterial toxoids and keyhole limpet haemocyanin.
  • More recently developed carriers which induce T-cell help in the immune response include the hepatitis-B core antigen (also called the nucleocapsid protein), presumed T-cell epitopes such as Thr-Ala-Ser-Gly-Val-Ala-Glu-Thr-Thr-Asn-Cys, beta-galactosidase and the 163-171 1 peptide of interleuldn-1.
  • the latter compound may variously be regarded as a carrier or as an adjuvant or as both.
  • several copies of the same or different peptides of the invention may be cross-linked to one another; in this situation there is no separate carrier as such, but a carrier function may be provided by such cross-linking.
  • Suitable cross-linking agents include those listed as such in the Sigma and Pierce catalogues, for example glutaraldehyde, carbodiimide and succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate, the latter agent exploiting the -SH group on the C-terminal cysteine residue (if present).
  • the peptide is prepared by expression of a suitable nucleotide sequence in a suitable host, then it may be advantageous to express the peptide as a fusion product with a peptide sequence which acts as a carrier. Kabigen's "Ecosec" system is an example of such an arrangement.
  • the peptide of the invention may be linked to other antigens to provide a dual effect.
  • antibody-like molecules may be useful in the practice of the invention including, for example, antibody fragments or derivatives which retain their antigen-binding sites, synthetic antibody-like molecules such as single-chain Fv fragments (ScFv) and domain antibodies (dAbs), and other molecules with antibody-like antigen binding motifs.
  • ScFv single-chain Fv fragments
  • dAbs domain antibodies
  • antibody-like molecules are included by the term antibody as used herein.
  • peptidomimetic compounds may also be useful in the practice of the invention.
  • polypeptide or “peptide” we include not only molecules in which amino acid residues are joined by peptide (-CO-NH-) linkages but also molecules in which the peptide bond is reversed
  • Such retro-inverso peptidomimetics may be made using methods known in the art, for example such as those described in Mézière et al (1997) J. Immunol. 159, 3230-3237 . This approach involves making pseudopeptides containing changes involving the backbone, and not the orientation of side chains. Mézière et al (1997) show that, at least for MHC class II and T helper cell responses, these pseudopeptides are useful.
  • Retro-inverse peptides which contain NH-CO bonds instead of CO-NH peptide bonds, are much more resistant to proteolysis.
  • the peptide bond may be dispensed with altogether provided that an appropriate linker moiety which retains the spacing between the C ⁇ atoms of the amino acid residues is used; it is particularly preferred if the linker moiety has substantially the same charge distribution and substantially the same planarity as a peptide bond.
  • the peptide may conveniently be blocked at its N- or C-terminus so as to help reduce susceptibility to exoproteolytic digestion.
  • a derivative or fusion of a polypeptide of the invention or variant, fragment or fusion thereof which may be particularly useful may comprise the polypeptide of the invention or variant, fragment or fusion therof and a further portion.
  • Said further portion may confer a desirable feature on the said molecule; for example, the portion may useful in detecting or isolating the molecule, or promoting cellular uptake of the molecule.
  • the portion may be, for example, a radioactive moiety, a fluorescent moiety, for example a small fluorophore or a green fluorescent protein (GFP) fluorophore, as well known to those skilled in the art.
  • GFP green fluorescent protein
  • the moiety may be an immunogenic tag, for example a Myc, FLAG or HA (haemagglutinin) tag, as known to those skilled in the art or may be a lipophilic molecule or polypeptide domain that is capable of promoting cellular uptake of the molecule, as known to those skilled in the art, for example as characterised for a Drosophila polypeptide (see, for example, Derossi et al (1998) Trends Cell Biol 8, 84-87 ).
  • Further useful tags include a tag that is capable of being phosphorylated, for example a tag capable of being phosphorylated by protein kinase A. Such a tag may be useful in introducing a radioactive label, for example 32 P or 33 P, onto the polypeptide.
  • Compounds, identifiable in the screening method, which mimic the effect of a particular phosphoinositide on a polypeptide, for example TAPP are believed to be useful in treating diabetes and/or other conditions, as indicated above.
  • Compounds identifiable in the screening methods of the invention that inhibit binding of a phosphoinositide to the said polypeptide are believed to be useful in treating cancer.
  • Compounds may be useful, for example, in the treatment of diabetes by switching on insulin-stimulated signal transduction pathways or in the treatment of cancer by inhibiting cell proliferation or promoting apoptosis.
  • Compounds may also be useful in the modulation or resolution of inflammation or platelet activation, as discussed above.
  • certain compounds found in the screening methods may be able to enhance cell proliferation in a beneficial way and may be useful, for example in the regeneration of nerves or in wound healing.
  • the invention further provides a compound capable of altering the expression of a polypeptide of the invention, for example TAPP.
  • the said compound may be an antisense molecule or ribozyme directed (for example, capable of binding to a polynucleotide encoding TAPP under physiological conditions) against a polynucleotide encoding a polypeptide of the invention, for example TAPP.
  • the nucleic acid of the invention may be an antisense oligonucleotide, for example an antisense oligonucleotide directed against a nucleic acid encoding a polypeptide of the invention such as the human TAPP gene.
  • Antisense oligonucleotides are single-stranded nucleic acid, which can specifically bind to a complementary nucleic acid sequence. By binding to the appropriate target sequence, an RNA-RNA, a DNA-DNA, or RNA-DNA duplex is formed. These nucleic acids are often termed "antisense" because they are complementary to the sense or coding strand of the gene.
  • oligonucleotide is bound to a DNA duplex. It was found that oligonucleotides could recognise sequences in the major groove of the DNA double helix. A triple helix was formed thereby. This suggests that it is possible to synthesise a sequence-specific molecules which specifically bind double-stranded DNA via recognition of major groove hydrogen binding sites.
  • the nucleic acid of the invention may be an antisense oligonucleotide, for example an antisense oligonucleotide directed against a nucleic acid encoding a polypeptide of the invention such as the human TAPP gene.
  • Antisense oligonucleotides are single-stranded nucleic acid, which can specifically bind to a complementary nucleic acid sequence.
  • the above oligonucleotides can inhibit the function of the target nucleic acid. This could, for example, be a result of blocking the transcription, processing, poly(A)addition, replication, translation, or promoting inhibitory mechanisms of the cells, such as promoting RNA degradations.
  • Antisense oligonucleotides are prepared in the laboratory and then introduced into cells, for example by microinjection or uptake from the cell culture medium into the cells, or they are expressed in cells after transfection with plasmids or retroviruses or other vectors carrying an antisense gene.
  • Antisense oligonucleotides were first discovered to inhibit viral replication or expression in cell culture for Rous sarcoma virus, vesicular stomatitis virus, herpes simplex virus type 1, simian virus and influenza virus. Since then, inhibition of mRNA translation by antisense oligonucleotides has been studied extensively in cell-free systems including rabbit reticulocyte lysates and wheat germ extracts.
  • Oligonucleotides are subject to being degraded or inactivated by cellular endogenous nucleases.
  • modified oligonucleotides eg having altered internucleotide linkages, in which the naturally occurring phosphodiester linkages have been replaced with another linkage.
  • Agrawal et al (1988) Proc. Natl. Acad. Sci. USA 85, 7079-7083 showed increased inhibition in tissue culture of HIV-1 using oligonucleotide phosphoramidates and phosphorothioates.
  • Oligonucleotides having artificial linkages have been shown to be resistant to degradation in viva.
  • Shaw et al (1991) in Nucleic Acids Res. 19, 747-750 report that otherwise unmodified oligonucleotides become more resistant to nucleases in vivo when they are blocked at the 3 ⁇ end by certain capping structures and that uncapped oligonucleotide phosphorothioates are not degraded in vivo.
  • oligonucleotide is a deoxyribonucleic acid (DNA), although ribonucleic acid (RNA) sequences may also be synthesised and applied.
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • the oligonucleotides useful in the invention preferably are designed to resist degradation by endogenous nucleolytic enzymes. In vivo degradation of oligonucleotides produces oligonucleotide breakdown products of reduced length. Such breakdown products are more likely to engage in non-specific hybridization and are less likely to be effective, relative to their full-length counterparts. Thus, it is desirable to use oligonucleotides that are resistant to degradation in the body and which are able to reach the targeted cells.
  • the present oligonucleotides can be rendered more resistant to degradation in vivo by substituting one or more internal artificial internucleotide linkages for the native phosphodiester linkages, for example, by replacing phosphate with sulphur in the linkage.
  • linkages examples include phosphorothioates, methylphosphonates, sulphone, sulphate, ketyl, phosphorodithioates, various phosphoramidates, phosphate esters, bridged phosphorothioates and bridged phosphoramidates.
  • Such examples are illustrative, rather than limiting, since other internucleotide linkages are known in the art. See, for example, Cohen, (1990) Trends in Biotechnology .
  • oligonucleotides having one or more of these linkages substituted for the phosphodiester internucleotide linkages is well known in the art, including synthetic pathways for producing oligonucleotides having mixed internucleotide linkages.
  • Oligonucleotides can be made resistant to extension by endogenous enzymes by "capping" or incorporating similar groups on the 5' or 3' terminal nucleotides.
  • a reagent for capping is commercially available as Amino-Link II TM from Applied BioSystems Inc, Foster City, CA. Methods for capping are described, for example, by Shaw et al (1991) Nucleic Acids Res. 19, 747-750 and Agrawal et al (1991) Proc. Natl. Acad. Sci. USA 88(17), 7595-7599 .
  • oligonucleotides resistant to nuclease attack are for them to be "self-stabilised” as described by Tang et al (1993) Nucl. Acids Res. 21, 2729-2735 .
  • Self-stabilised oligonucleotides have hairpin loop structures at their 3' ends, and show increased resistance to degradation by snake venom phosphodiesterase, DNA polymerase. I and fetal bovine serum.
  • the self-stabilised region of the oligonucleotide does not interfere in hybridization with complementary nucleic acids, and pharmacokinetic and stability studies in mice have shown increased in vivo persistence of self-stabilised oligonucleotides with respect to their linear counterparts.
  • nucleic acid has sufficient nucleotide sequence similarity with the said human nucleic acid that it can hybridise under moderately or highly stringent conditions, as discussed above.
  • stringency of nucleic acid hybridization depends on factors such as length of nucleic acid over which hybridisation occurs, degree of identity of the hybridizing sequences and on factors such as temperature, ionic strength and CG or AT content of the sequence.
  • any nucleic acid which is capable of selectively hybridising as said is useful in the practice of the invention.
  • Nucleic acids which can selectively hybridise to the said human nucleic acid include nucleic acids which have >95% sequence identity, preferably those with >98%, more preferably those with >99% sequence identity, over at least a portion of the nucleic acid with the said human nucleic acid.
  • human genes usually contain introns such that, for example, a mRNA or cDNA derived from a gene would not match perfectly along its entire length with the said human genomic DNA but would nevertheless be a nucleic acid capable of selectively hybridising to the said human DNA.
  • the nucleic acid capable of selectively hybridising to the said human nucleic acid such as mRNA and which is used in the methods of the invention further comprises a detectable label.
  • detecttable label any convenient radioactive label such as 32 P, 33 P or 35 S which can readily be incorporated into a nucleic acid molecule using well known methods; any convenient fluorescent or chemiluminescent label which can readily be incorporated into a nucleic acid is also included.
  • detecttable label also includes a moiety which can be detected by virtue of binding to another moiety (such as biotin which can be detected by binding to streptavidin); and a moiety, such as an enzyme, which can be detected by virtue of its ability to convert a colourless compound into a coloured compound, or vice versa (for example, alkaline phosphatase can convert colourless o-nitrophenylphosphate into coloured o-nitrophenol).
  • PCR polymerase chain reaction
  • chimeric monoclonal antibodies can be produced using genetic constructs derived from hybridoma cells producing the monoclonal antibodies described above. Methods for producing chimeric antibodies are known in the art. See, for review, Morrison, Science 229:1202 (1985 ); Oi et al, BioTechniques 4:214 (1986 ); Cabilly et al., U.S. Patent No.
  • Typical techniques for binding the above-described labels to antibodies are provided by Kennedy et al., Clin. Chim. Acta 70:1-31 (1976 ), and Schurs et al, Clin. Chim. Acta 81:1-40 (1977 ). Coupling techniques mentioned in the latter are the glutaraldehyde method, the periodate method, the dimaleintide method, the m-maleimidobenzyl-N-hydroxy-succinimide ester method.
  • Example 1 Identification of PH domains with novel phosphoinositide binding specificities.
  • the second messsenger phospatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P 3 ) is generated by the action of phosphatidylinositol 3-kinase (PI 3-kinase) and regulates a plethora of cellular processes.
  • An approach for dissecting the mechanisms by which these processes are regulated, is to identify proteins that interact specifically with PtdIns(3,4,5)P 3 .
  • the pleckstrin homology (PH) domain has become recognised as the specialised module used by many proteins to interact with PtdIns(3,4,5)P 3 .
  • TAPP1 PtdIns(3,4)P 2
  • PtdIns3P PEPP1 & AtPH1 and also PEPP2 and PEPP3
  • PtdIns4P FAPP1
  • Ptdlns(3,5)P 2 Centaurin- ⁇ 2
  • ARF ADP ribosylation factor
  • DAPP dual adaptor for phosphotyrosine and 3-phosphoinositides
  • EST expressed sequence tag
  • FAPP1 PtdIns- F our-phosphate A da P tor P rotein-1
  • GAP GTPase activating protein
  • GST glutathione-S-transferase
  • NCBI National Center for Biotechnology Information
  • PKC protein kinase C
  • PDZ postsynaptic density protein ( P SD-95)/Drosophila disc large tumour suppressor ( D lg)/tight junction protein ( Z O1)
  • PDK 3-phosphoinositide-dependent protein kinase-1
  • PH pleckstrin homology
  • PEPP1 P tdIns-thr E e- P hosphate binding P H domain Protein-1
  • PI 3-kinase phosphoinositide 3-kinase
  • PKB protein kinase B
  • PP protein
  • Hybond-C extra was from Amersham Pharmacia Biotech, High Fidelity PCR kit from Roche, Human tissue (Catalogue number 7780-1), mouse tissue (Catalogue number 7762-1) and human cancer cell line (Catalogue number 7757-1) Multiple Tissue Northern Blots from Clontech, Human Universal cDNA Library was from Strategene, pCR 2.1Topo vector and precast SDS polyacrylamide gels were from InVitrogen.
  • DAPP1 and Grpl [8] were expressed as fusion proteins with glutathione-S-transferase (GST) in 293 cells [4].
  • GST glutathione-S-transferase
  • Buffer A 50 mM Tris-HCl pH 7.5, 1 mM EGTA, 1 mM EDTA, 1% (by mass) Triton-X 100, 1 mM sodium orthovanadate, 50 mM sodium fluoride, 5 mM sodium pyrophosphate, 0.27 M sucrose, 1 ⁇ M microcystin-LR, 0.1% (by vol) ⁇ -mercaptoethanol and 'complete' proteinase inhibitor cocktail (one tablet per 50 ml, Roche).
  • Buffer B 50 mM Tris/HCl pH 7.5, 0.1 mM EGTA, 10 mM ⁇ -mercaptoethanol and 0.27M sucrose.
  • each protein that was amplified using the indicated EST as template was as follows: human TAPP1 (residues 95 to 404, accession number AI216176), mouse TAPP2 (residues 174 to 425, accession number AA111410), human FAPP1 (residues 1 to 99, accession number W32183), Arabidopsis thaliana AtPH1 (full length protein, residues 1 to 145, accession number, T04439), human PEPP1 (sequence in Fig 6 Ser-Ala-Ser to Arg-Pro-Gln, accession number N31123), mouse centaurin- ⁇ 2 (residues 266 to 390, accession number AA967911), putative human homologue of rat LL5 ⁇ (sequence Ser-Glu-Ser-Ala to Gln-Phe-Met-Asn, accession number AA863428), putative human isoform of LL5 ⁇ which we have termed T
  • PCR products were resolved on 1% agarose, gel purified, cloned into the pCR2.1 TOPO vector, sequenced and subcloned into the E.coli pGEX-4T-1 expression vector or the mammalian pEBG2T vector that codes for the expression of these proteins with a GST tag at the N-terminus.
  • the pGEX-4T-1 constructs encoding the PH domains of FAPP1, AtPH1, PEPP1, LL5 ⁇ , IL5 ⁇ , evectin-2 and PH30 were transformed into BL21 E. coli cells and a 0.5L culture was grown at 37 °C in Luria Broth containing 100 ⁇ g/ml ampicillin, until the absorbance at 600 nm was 0.6. 250 ⁇ M isopropyl- ⁇ -D-galactosidase was added and the cells cultured for a further 16 h at 26 °C.
  • the cells were resuspended in 25 ml of ice-cold Buffer A and lysed by one round of freeze thawing and the lysates sonicated to fragment the DNA.
  • the lysates were centrifuged at 4°C for 30 min at 20, 000 x g, the supernatant filtered through a 0.44 micron filter and incubated for 60 min on a rotating platform with 1 ml of glutathione-Sepharose previously equilibrated in Buffer A.
  • the suspension was centrifuged for 1 min at 3000 x g, the beads washed three times with 15 ml of Buffer A containing 0.5 M NaCl, and then a further ten times with 15 ml of Buffer B.
  • the protein was eluted from the resin at ambient temperature by incubation with 2 ml of Buffer B containing 20 mM glutathione, and the beads removed by filtration through a 0.44 micron filter. The eluate was divided into aliquots, snap frozen in liquid nitrogen, and stored at -80°C.
  • the cells were lysed in 1 ml of ice-cold Buffer A, the lysates pooled, centrifuged at 4°C for 10 min at 13, 000 x g and the GST-fusion proteins were purified by affinity chromatography on glutathione-Sepharose and stored as described above.
  • TAPP1, TAPP2 , FAPP1 and PEPP Cloning TAPP1, TAPP2 , FAPP1 and PEPP1.
  • Full length human TAPP1, full length mouse TAPP2, partial mouse TAPP1, partial human TAPP2, and full length human and mouse FAPP1 sequences were deduced by sequencing the EST clones listed in Table 3.
  • Several EST clones possessed identical sequences, and had the same in-frame stop codon 5' to the predicted initiating ATG codon and possessed a stop codon at the same position at the 3' end of the gene.
  • the constructs used to express full length and deletion mutants of TAPP1 and TAPP2 were generated by PCR, using as a template ESTs encoding full length human TAPP1 (accession number AI216176) and full length mouse TAPP2 (accession number AA111410).
  • the PCR primers used were designed to incorporate a Kozak site, and an initiating ATG codon followed by a Flag epitope tag and the resulting PCR product
  • PEPP1 and FAPP1 Cloning of PEPP1 and FAPP1.
  • a Stratagene Human Universal cDNA Library was screened with a DNA probe corresponding to the PH domains of PEPP1 and FAPP1 and we were able to isolate a clone encoding each of these proteins using this approach.
  • the partial sequence of PEPP1 that contains the 5'end of the coding sequence was obtained by sequencing of ESTs with NCBI accession numbers N49341and N31123.
  • PEPP1 To obtain a full length cDNA encoding PEPP1, we screened a Stratagene Human Universal cDNA Library with a DNA probe corresponding to the N-terminal 15 to 169 residues of PEPP1 and we isolated a full length PEPP1 cDNA which had a stop codon 5' to the predicted initiating ATG codon an open reading frame encoding 779 amino acids followed by a stop codon. Interrogation of the EST databases with the full length PEPP1 sequ4ence identified 2 closely related isoforms of this protein termed PEPP2 and PEPP3.
  • PEPP2 The sequence of human PEPP2 was deduced by sequencing the following EST clones: A1808805 (kidney), AA232124(brain), W91917 (foetal liver and spleen) and AI638629 (germ cell line). The sequence of PEPP2 is likely to be full length as there is a stop codon 5' to the predicted initiating ATG codon. ESTs relating to PEPP3 are AI739438, BE303674 and F23241.
  • cDNA corresponding to full length human TAPP1, partial human TAPP2 (residues 18 to 304), partial human PEPP1 (residues encoding sequence Ser-Ala-Ser to Arg-Pro-Gln), partial human PEPP2 (residues 154 to 654) and mouse partial mouse centaurin- ⁇ 2 (residues 266 to 390) were 32 P-labelled by random priming using a multi-prime DNA labelling kit (Amersham Pharmacia). These probes were then used to screen Northern blots using Rapid-Hyb Buffer (Amersham Pharmacia) according to the protocol provided by the manufacturer.
  • Protein-Lipid overlay To assess the phosphoinositide binding properties of each PH domain, a protein-lipid overlay assay was performed using the GST fusion proteins as described previously [4, 15]. Briefly, 1 ⁇ l of lipid solution containing 1-100 pmol of phospholipids dissolved in a mixture of choroform:methanol:water (1:2:0.8) was spotted onto Hybond-C extra membrane and allowed to dry at room temperature for 1 h. The membrane was blocked in 3% (by mass) fatty acid-free BSA in TBST (50 mM Tris/HCl pH 7.5, 150 mM NaCl and 0.1% Tween-20 (by vol) for 1h.
  • TBST 50 mM Tris/HCl pH 7.5, 150 mM NaCl and 0.1% Tween-20 (by vol) for 1h.
  • the membrane was then incubated overnight at 4 °C with gentle agitation in the same solution containing 0.2 ⁇ g/ml of the indicated GST fusion protein.
  • the membranes were washed 6 times over 30 min in TBST and then incubated for lh with 1/1000 dilution of anti-GST monoclonal antibody (Sigma).
  • the membranes were washed as before, then incubated for 1h with 1/5000 dilution of anti-mouse-HRP conjugate (Pierce).
  • the membranes were washed 12 times over lh in TBST and the GST-fusion protein bound to the membrane by virtue of its interaction with phospholipid was detected by enhanced chemiluminescence.
  • TAPP1 and TAPP2 bind specifically to PtdIns(3,4)P 2 .
  • Two of the novel sequences identified encoded related proteins which were termed TAPP1 and TAPP2 (Table 1). Clones encoding the full length human TAPP1 (accession number AF286160) and mouse TAPP2 (accession number AF286161) as well as a partial mouse TAPP1 (accession number AF286165) and human TAPP2 (accession number AF286164), were isolated as described in the Methods section.
  • Human TAPP1 is a protein of 404 amino acids and mouse TAPP2 is a protein of 425 amino acids ( Fig 3A ).
  • TAPP1 and TAPP2 A stop codon immediately 5' to the predicted initiating ATG codon indicates that both human and mouse TAPP1 and TAPP2 protein sequences are full length.
  • Analysis of the TAPP1 and TAPP2 sequences revealed the presence in each protein of two PH domains, of which only the C-terminal PH domain possesses the PPBM ( Fig 3A ). Hence these proteins were termed TAPP for TA ndem P H domain containing P rotein.
  • the amino acid sequences of TAPP1 and TAPP2 are 58% identical over the first 300 amino acids, which encompasses both of the PH domains. There is little homology between the C-terminal 100 residues of TAPP1 and TAPP2, except that 7 out of the 11 C-terminal amino acids of TAPP1 and TAPP2 are identical.
  • TAPP1 and TAPP2 conform to the minimal sequence motif (Ser/Thr-Xaa-Val/Ile [22, 23]) required for binding to a PDZ domain.
  • Ser/Thr-Xaa-Val/Ile [22, 23] a binding site for an SH3 domain
  • no other known catalytic domains are present.
  • Interrogation of the NCBI human genome database with the TAPP1 sequence indicated that it is located on chromosome 10q25.3-q26.2. Although the genomic fragment that encompases TAPP2 (accession number AC067817) has been sequenced, its chromosomal location is not yet known.
  • the isolated C-terminal PH domains of TAPP1 and TAPP2 (which possess the PPBM), when expressed as GST-fusion proteins, interacted with PtdIns(3,4)P 2 but did not bind to PtdIns(3,4,5)P 3 or any other phosphoinositides tested ( Fig 2 ).
  • Surface plasmon resonance studies indicated that the isolated C-terminal PH domain of TAPP1 and TAPP2 interacted with PtdIns(3,4)P 2 with apparent Kd values of 5 nM and 30 nM, respectively (Table 2).
  • the N-terminal PH domain of TAPP1 and TAPP2 failed to interact with any phosphoinositide tested ( Fig 4A and Table 2).
  • TAPP1 and TAPP2 mRNA were investigated by Northern blot analysis.
  • TAPP1 was detected as a 4 kb transcript in all tissues examined with the highest levels observed in skeletal muscle, spleen, lung, thymus and placenta ( Fig 3B ).
  • TAPP2 was detected as a 6 kb transcript in all tissues examined with the highest levels observed in heart and kidney ( Fig 3B ).
  • FAPP1 is a specific PtdIns4P binding protein.
  • the identified PH domain termed FAPP1 (Table 1), possessing Gln instead of Lys or Arg at the third conserved residue of the PPBM, exhibited a high affinity for PtdIns4P (K d 20 nM), but did not bind to any other phosphoinositide ( Fig 2 & Table2).
  • the full length human and mouse FAPP1 sequences ( Fig 5 ) were deduced from the sequencing of ESTs listed in Table 3.
  • Human FAPP1 encodes a protein of 300 amino acids and a stop codon immediately 5' to the predicted initiating ATG codon indicates that both the human and mouse FAPP1 protein sequences are full length.
  • FAPP1 Interrogation of the human genome NCBI database indicated that the FAPP1 gene was located on an unmapped region of chromosome 2 (accession number NT_003398). Analysis of the FAPP1 sequence revealed the presence of an N-terminal PH domain and a proline rich region located towards the C-terminus that could mediate binding to SH3 domains ( Fig 5 ). FAPP1 is likely to be expressed widely, because 27 EST clones encoding this protein were derived from several tissues (Table 3). However, FAPP1 may not be an abundant transcript as we were unable to detect significant levels of FAPP1 mRNA expression in any tissue or cell line examined (data not shown).
  • FAPP2 also binds specifically to PtdIns4P.
  • AtPH1 and mammalian PEPP bind PtdIns3P specifically.
  • AtPH1 is a small 145 residue Arabidopsis protein, whose physiological role is unknown. It consists of one PH domain with a short N-terminal extension and is expressed in all plant tissues [24].
  • PEPP1 is a novel mammalian protein, whose partial sequence ( Fig 6A ) and full length sequence ( Fig 8A ) we have deduced from sequencing of several ESTs (Table 3). The partial sequence is likely to comprise the N-terminal end of PEPP1 as there is an in-frame stop codon 5' to the predicted initiating ATG codon.
  • the PH domain of PEPP1 is located at the N-terminal region of PEPP1. There are also 2 proline rich regions that could comprise SH3 binding sites. Analysis of the NCBI human genome database shows that the PEPP1 gene is located on an unmapped region of chromosome 19 (accession number AC026803).
  • PEPP1 mRNA was expressed at very high levels in a melanoma cancer cell line as a 3 kb fragment, but was not significantly expressed in the other 7 non-melanoma cancer cell lines that were investigated ( Fig 6B ).
  • PEPP1 may be selectively expressed in melanoma or melanocytes.
  • Table 3 the three human EST clones encoding PEPP1 that we have identified thus far are derived from either a melanoma or a melanocyte cDNA library (Table 3).
  • PEPP2 and PEPP3 Interrogation of the NCBI database with the PEPP1 sequence revealed 2 other proteins that appear to be related isoforms of PEPP1 termed PEPP2 and PEPP3. The identity between these proteins is most notable in the PH domain, especially in the region that encompasses the PPBM as well as a region of 30 amino acids that precedes the PH domain. PEPP1, PEPP2 and PEPP3 are poorly conserved in the region C-terminal to the PH domain ( Fig 8A ). PEPP2, but not PEPP1 or PEPP3 also possesses two WW domains ( Rotin (1998) Curr Top Microbiol Immunol 228, 115-133 ) in a region N-terminal to the PH domain ( Fig 8A ).
  • PEPP2 may be more widely expressed than PEPP1 as Northern Blot analysis shows that PEPP2 mRNA is present in high levels in heart and kidney and also expressed at a lower level in other tissues.
  • PEPP3 may not be an abundant transcript as we were unable to detect significant levles of PEPP3 mRNA expression in any tissue or cell line examined (data not shown).
  • the four PEPP3 ESTs that are present in the database are derived from brain, colon, mammary gland and skeletal muscle (see methods). PEPP2 and PEPP3 are also considered to bind PtdIns3P.
  • Centaurin- ⁇ 2 is a PtdIns(3,5)P 2 binding protein.
  • Human centaurin- ⁇ 2 is an uncharacterised 778 amino acid protein (cloned by T. Jackson and colleagues, University College London, accession number CAB41450), possessing a PH domain (residues 267-363) followed by a putative ARF GAP domain (residues 399-520) and three ankyrin repeats at its C-terminus.
  • the PH domains of both mouse and human centaurin- ⁇ 2 possess Asn instead of a Lys or Arg at the third conserved residue of the PPBM (Table 1).
  • centaurin- ⁇ 2 The PH domain of mouse centaurin- ⁇ 2 exhibited moderate affinity for PtdIns(3,5)P 2 but did not bind to any other phosphoinositide tested ( Fig 2 ). Centaurin- ⁇ 2 is likely to be a widely expressed protein as 12 EST clones encoding it were derived from several tissues and Northern blot analysis indicated that mouse centaurin- ⁇ 2 was expressed as a 4.5 kb fragment in all tissues investigated (data not shown).
  • PtdIns(3,4)P 2 regulates the same physiological processes as PtdIns(3,4,5)P 3 , as it is formed as a breakdown product of PtdIns(3,4,5)P 3 and many of the PH domains that interact with PtdIns(3,4,5)P 3 also bind to PtdIns(3,4)P 2 (as discussed in the introductory section above).
  • TAPP1 and TAPP2 are the first proteins to be identified that interact with PtdIns(3,4)P 2 specifically and may therefore be key mediators of cellular responses that are regulated specifically by this second messenger.
  • TAPP1 and TAPP2 are ESTs encoding a TAPP1 homologue derived from zebrafish and chicken (Table 3). Further studies are required to characterise the physiological role of TAPP1 and TAPP2, but it is possible that they function as adaptor proteins to recruit proteins that interact with them to cellular membranes in response to extracellular signals that lead to the generation of PtdIns(3,4)P 2 . However, it is possible that the in vitro lipid binding properties of TAPP1 and TAPP2, as well as the other PH domain containing proteins that we have characterised in this study, could differ from their in vivo binding specificities.
  • the inositol polyphosphate head groups of the phosphoinositides could be the natural ligands for these proteins.
  • TAPP1 and TAPP2 could also potentially interact with proteins containing PDZ domains through their C-terminal Ser-Xaa-Val residues and TAPP2 could bind to SH3 domains through two proline rich motifs located towards its C-terminus.
  • PtdIns 4P the only PH domain previously shown to bind PtdIns4P with some specificity is derived from a plant PtdIns 4-kinase which also interacts weakly with PtdIns(4,5)P 2 [28].
  • FAPP1 Fig 5
  • PtdIns 4P only binds PtdIns 4P and does not interact with PtdIns(4,5)P 2 ( Fig 2 , Table 2).
  • a key role of PtdIns 4P in mammalian cells is to act as an intermediate in the synthesis of PtdIns(4,5)P 2 .
  • FAPP1 does not possess a catalytic domain that would indicate a role in regulating the synthesis or breakdown of PtdIns4P in cells.
  • PtdIns3P plays an important role in regulating golgi to vacuole or lysosome membrane trafficking as well as endosome function [29].
  • Several proteins (e.g. EEA1) regulating these processes have been found to interact with PtdIns3P through a particular type of Zinc finger domain (known as the FYVE domain) [30].
  • the only other PH domain-containing protein other than PEPP1 and AtPH1 previously reported to interact with PtdIns3P is phospholipase C ⁇ 1[31].
  • phospholipase C ⁇ 1 may be less specific for PtdIns3P than PEPP1 and AtPH1, as it also possessed significant affinity for PtdIns(4,5)P 2 and PtdIns(3,4,5)P 3 [31].
  • the evidence indicates that phospholipase C ⁇ 1 may be recruited to plasma membranes through an interaction of its PH domain with both PtdIns 3P (or other phosphoinositide) and the G ⁇ regulatory subunits [31, 32].
  • PEPP1 A potentially interesting feature of PEPP1, is that its expression may be restricted to melanoma and or melanocytes as Northern blot analysis indicated that PEPP1 was expressed at very high levels in a melanoma cell line, but not in 7 other non-melanoma cancer cell lines or 12 tissues that were investigated ( Fig 6B ). Further work is required to determine whether PEPP1 expression is elevated in all melanoma cells compared to normal melanocytes. It is interesting that a closely related homologue of PEPP1, termed PEPP2, appears to be more widely expressed ( Fig 8B ). PEPP2 and PEPP3 possess a very similar sequence surrounding the PPBM of their PH domains indicating that they may also interact with PtdIns3P.
  • Plant cells contain high levels of PtdIns3P as well as PtdIns(3,4)P 2 but no PtdIns(3,4,5)P 3 has been detected [33], consistent with the apparent lack of Class 1A PI 3-kinases in plants.
  • AtPH1 is the first plant protein that has been shown to interact with PtdIns3P and may play an important role as an adaptor protein in regulating signalling processes in plants that are mediated by PtdIns3P. There are no apparent homologues of PEPP1 or AtPH1 in Drosophila, C.elegans or S. cerevisiae.
  • centaurins The ARF family of GTP binding proteins regulate membrane trafficking and the actin cytoskeleton [34].
  • a family of ARF GAP proteins collectively termed centaurins, have been identified and all possess one or more PH domains and an ARF GAP catalytic domain [35].
  • the PH domain on centaurin- ⁇ 1 interacts with PtdIns(3,4,5)P 3 and centaurin- ⁇ 1 is recruited to cell membranes after PI 3-kinase is activated [7].
  • centaurin- ⁇ 4 has been shown to be activated by the interaction of its PH domain with PtdIns(4,5)P 2 and, in contrast to centaurin- ⁇ 1, does not bind to PtdIns(3,4,5)P 3 [36].
  • centaurin- ⁇ 2 interacts with PtdIns(3,5)P 2 , albeit with moderate affinity, suggests that centaurin- ⁇ 2 may be regulated by this lipid. Further investigation is required to establish whether PtdIns(3,5)P 2 can lead to the activation of centaurin- ⁇ 2. No protein has previously been shown to interact specifically with PtdIns(3,5)P 2 and the physiological processes regulated by this lipid are not known.
  • yeast PtdIns(3,5)P 2 is generated in response to osmotic stress [37] by phosphorylation of PtdIns3P at the D5 position by a kinase termed Fab1[38, 39].
  • Fab1[38, 39] a kinase
  • this Example describes a group of novel PH domain containing proteins that possess interesting phosphoinositide binding specificities.
  • TAPP1, TAPP2, FAPP1 and AtPH1 may function as adaptor molecules as they possess no obvious catalytic moieties.
  • the physiological processes that are regulated by the PH domain-containing proteins described in this paper it may not only be important to knock out these proteins in cells and mice but also to identify the proteins that they interact with.
  • Polypeptides interacting with TAPP1, TAPP2, PEPP1, PEPP2, PEPP3 or FAPP are identified using yeast two hybrid methods and/or immunoprecipitation/coprecipitation methods.
  • the methods are performed on stimulated and unstimulated cells; polypeptides that interact with TAPP1, TAPP2, PEPP1, PEPP2, PEPP3 or FAPP (for example FAPP1 or FAPP2) in one cell state only (or to different extents in the different cell states) are of particular interest.
  • the methods may also be performed (for comparison) with mutated TAPP1, TAPP2, PEPP1, PEPP2, PEPP3 or FAPP polypeptides, for example mutants which do not bind the relevant phosphoinositide.
  • Coprecipitated polypeptides are analysed by microsequencing and mass spectrometry.
  • the amino acid sequence information is used to identify/isolate polynucleotides encoding the amino acid sequence, using standard molecular biology techniques.
  • Particular enzymes such as particular lipid phosphatases or inositol lipid kinases, may be assayed using the PH domains described herein, for example using TAPP1, TAPP2, PEPP1, PEPP2, PEPP3 or FAPP (for example FAPP1 or FAPP2).
  • the assay system makes use of the ability of the PH domains to bind specifically to PtdIns(3,4)P 2 , PtdIns3P, PtdIns4P or PtdIns(3,5)P 2 but not capable of binding to PtdIns(3,4,5)P 3 , when the phosphoinositide is the product (or substrate) of a lipid kinase or phosphatase reaction.
  • the PH domain may be used as a recombinant protein fused to a reporter tag such as a green fluorescent protein or labelled with a fluorescent chromophore.
  • a Class II PI3 kinase may generate PtdIns3P, which may be measured using PEPP or AtPH1.
  • a PI4 kinase generates PtdIns4P, which may be measured using FAPP.
  • Fablp[38, 39] generate Ptd(3,5)P 2 , which may be measured using centaurin- ⁇ 2.
  • changes in the substrate for an enzyme may be measured.
  • Fablp converts PtdIns3P to PtdIns(3,5)P 2 and a PH domain which binds to PtdIns3P (for example the PH domain of PEPP1 or AtPH1) may be used to monitor the level of PtdIns3P and thereby Fablp activity.
  • the group of 5' phosphatases target PtdIns(3,4,5)P 3 and also PtdIns4,5P 2 , to yield PtdIns4P.
  • FAPP may be used in measuring such 5' phosphatase activity.
  • FAPP may also be useful in monitoring a 4' phosphatase, for example Saclp from yeast and homologues thereof, which appears to be specific for dephosphorylating PtdIns4P to phosphoinositide (see, for example, Hughes et al (2000) Biochem J 350(2), 337-352 ; Nemoto et al (2000) J Biol Chem 275(44), 34293-24305 (rat homologue); Hughes et al (2000) J Biol Chem 275(2), 801-808 ).
  • a FRET (fluorescence resonance energy transfer) system may be used.
  • a soliud phase assay with the substrate lipid bound to the surface of a microtitre plate may be used.
  • PH domain binding to the product formed in the immobilised lipid layer is detected by time resolved FRET.
  • substrate lipids in a lipid layer incorporating a donor chromophore immobilised in wells of a 96 well microtitre plate are incubated with the appropriate enzyme (or sample to be tested for the appropriate enzyme) in the presence of the appropriate recombinant PH domain fused to green fluorescent protein (GFP; including mutant GFPs, as discussed above) and ATP.
  • GFP green fluorescent protein
  • the PH-GFP binds specifically to the product (or in an alternative, the substrate) and in doing so is brought into close enough proximity with the chromophore in the lipid layer for FRET to occur. This may be detected using methods well known to those skilled in the art.
  • This system does not use radioisotopes; does not require separation of reaction products, allowing the system to be used in high throughput screens; does not use lipid vesicles, thereby reducing "false positives" in inhibitor screens due to vesicle disruption by the test compound; and may be used for several enzymes, depending on the lipid and PH domains chosen.
  • the system may be used for making real time measurements throughout the curse of the reaction.
  • Other methods for example using radioisotopes
  • the PH domain may be "tagged" in other ways, for example with an alternative chromophore, an epitope tag or a detectable enzyme, as well known in interaction assays, for example immunoassays.
  • the PH domain may be in the form of a GST fusion protein labelled with a terbium chelate (Terbium Lance Chelate, LKB Wallac) as energy donor and rhodamine labeleld phosphatidylethanolamine as energy acceptor.
  • a terbium chelate Tebium Lance Chelate, LKB Wallac
  • the intrinsic fluorescence of tryptophan residues in the PH domain may change on binding to the phosphoinositide, and this may be used in monitoring the binding of the PH domain to the phosphoinositide, and thereby determining the amount of phosphoinositide present.
  • the assay configuration may consist of a microtitre plate coated with a mixture containing the substrate phosphoinositide, for example 0.8nmols, phosphatidylserine, 0.7nmols, and rhodamine labelled phosphatidylethanolamine, 01.5nmmols, giving a total of 2nmols lipid per well.
  • the PH-GST terbium chelate is used at a concentration of 0.175 ⁇ g/ml in a final volume of 50 ⁇ l.
  • a well may be "spiked" with the product lipid at various concentrations.
  • the labelled PH domain is added to the plate and time resolved measurements of fluorescence are taken. For example, excitation at 340nm, emission at 601 nm and a time gate of 50 to 800 ⁇ sec may be used. Detection limits are in the low pmol range.
  • Enzyme activity can be determined by measuring fluroescence over time.
  • the enzyme or sample is added with ATP (for example 0.1mM ATP).
  • Data points may be the mean of measurements of several wells (for example eight) read at 30 second intervals over 30 minutes.
  • the assay may be run as a homogenous fluid phase assay with the substrate lipid either in free solution or as lipid vesicles.
  • the fluid phase assay relies on reaction product competing for binding in a preformed detection complex.
  • the complex may be formed, for example, between Europium lance chelate labelled GST-PH domain, biotinylated short chain phosphoinositide (for example C6 product phosphoinositide) and streptavidin labelled allophycocyanin (APC).
  • Enzyme activity is detected by the conversion of nonbiotinylated short chain substrate phosphoinositide to product phosphoinositide, which competes for binding with the GST-PH domain in the preformed complex, resulting in a decrease in the FRET signal.
  • the system may be tested by adding biotinylated synthetic short chain product to the assay system.
  • the assay may contain 1 ⁇ l APC (for example 0.01 to 100 ⁇ g, preferably 0.1 to 10 ⁇ g), 1 ⁇ l of the Europium labelled GST-PH domain (for example 0.01 to 100 ⁇ g, preferably 0.1 to 10 ⁇ g) and increasing concentrations (for example from 0 to 300 pmol) of the water soluble biotinylated short chain product phosphoinositide in a final volume of 50 ⁇ l.
  • An excitation wavelength of 340, emission wavelength of 665nm and cut-off of 630 nm may be used.
  • non-biotinylated product phosphoinositide produced from the substrate phosphoinositide competes for binding to the GST-PH domain, reducing the observed signal.
  • the system may be tested by addition of increasing amounts of non-biotinylated product phosphoinositide.
  • the biotinylated product phosphoinositide may be present at 0.5 ⁇ M (25pmol/assay).
  • a typical assay set-up may be as follows:
  • the rate of decrease of time resolved FRET may be measured over 30 minutes at 30 sec intervals over a range of substrate phosphoinositide concentrations (for example 0 to 70 ⁇ M) and the initial rates estimated.
  • the interaction of the components of an assay may be detected using the Alpha ScreenTM bead system from BioSignal Packard (part of Packard Biscience), of 1744 rue William, Suite 600, Montreal, Quebec, Canada, H3J 1R4.

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Claims (18)

  1. Zusammensetzung mit einem Polypeptid, das sich spezifisch an PtdIns(3,4)P2 binden kann, sich aber nicht an PtdIns(3,4,5)P3 binden kann, wobei das Polypeptid eine PH-Domäne umfasst, die sich an PtdIns(3,4)P2 binden kann, sich aber nicht an PtdIns(3,4,5)P3 binden kann, wobei das Polypeptid die folgende Aminosäuresequenz umfasst:
    Figure imgb0017
    oder
    Figure imgb0018
    oder ein Fragment hiervon umfasst, wobei sich das Fragment spezifisch an PtdIns(3,4)P2 binden kann, sich aber nicht an PtdIns(3,4,5)P3 binden kann, oder wobei das Polypeptid eine Fusion eines genannten Fragments davon aufweist, wobei die Zusammensetzung einen Proteingehalt oder -inhalt hat, der wenigstens aus 50 Gew.-% des genannten Polypeptids besteht.
  2. Rekombinantes Polynucleotid, das zur Expression eines Polypeptids wie in Anspruch 1 definiert geeignet ist.
  3. Vektor, der zur Replikation in einer Säugetier-/eukaryotischen Zelle geeignet ist, mit einem Polynucleotid, das das Polypeptid wie in Anspruch 1 definiert kodiert.
  4. Polynucleotid oder Vektor nach Anspruch 2 oder 3, das/der keine Introns enthält.
  5. Wirtszelle, die ein rekombinantes Polynucleotid oder einen replizierbaren Vektor nach einem der Ansprüche 2 bis 4 umfasst.
  6. Verfahren zur Herstellung eines Polypeptids wie in Anspruch 1 definiert, wobei das Verfahren das Kultivieren einer Wirtszelle nach Anspruch 5, die das genannte Polypeptid exprimiert, und das Isolieren des genannten Polypeptids beinhaltet
  7. Verfahren nach Anspruch 6, wobei die genannte Wirtszelle eine eukaryotische Zelle ist.
  8. Antikörper, der gegenüber einem Polypeptid mit der folgenden Aminosäuresequenz reaktiv ist:
    Figure imgb0019
    oder
    Figure imgb0020
    wobei der Antikörper nicht mit einem anderen Polypeptid reagiert, das eine PH-Domäne umfasst.
  9. Verwendung eines Polypeptids nach Anspruch 1 in einem Screeningverfahren zum Identifizieren einer Verbindung oder Mischung, die zum Modulieren der Signalisierung durch PtdIns(3,4)P2 geeignet ist.
  10. Verwendung nach Anspruch 9, wobei das Verfahren die folgenden Schritte beinhaltet: (1) Aussetzen des genannten Polypeptids mit PtdIns(3,4)P2 in Anwesenheit einer Testverbindung oder -mischung; (2) Bestimmen, ob die Testverbindung oder -Mischung die Bindung von PtdIns(3,4)P2 an das genannte Polypeptid moduliert; und (3) Auswählen einer Verbindung oder Mischung, die die Bindung von PtdIns(3,4)P2 an das genannte Polypeptid moduliert.
  11. Verfahren zum Identifizieren einer Verbindung oder Mischung, die die Phospholipidbindungsaktivität eines Polypeptids wie in Anspruch 1 definiert moduliert, wobei das Verfahren Folgendes beinhaltet: Inkontaktbringen einer Verbindung oder Mischung mit dem genannten Polypeptid und Bestimmen, ob die Phospholipidbindungsaktivität des genannten Polypeptids in Anwesenheit der Verbindung oder Mischung im Vergleich zu der in Abwesenheit der genannten Verbindung oder Mischung geändert wird oder ist.
  12. Verfahren nach Anspruch 11, wobei die genannte Bindungsaktivität verringert wird oder ist.
  13. Verfahren nach Anspruch 11, wobei die genannte Bindungsaktivität erhöht wird oder ist.
  14. Verfahren nach einem der Ansprüche 11 bis 13, wobei das Verfahren in einer Zelle durchgeführt wird.
  15. Verfahren für den Nachweis und/oder die quantitative Bestimmung von PtdIns(3,4)P2 in einer Probe, wobei die Probe einem Polypeptid wie in Anspruch 1 definiert ausgesetzt wird und die Bindung des genannten Polypeptids an irgendein anwesendes PtdIns(3,4)P2 nachgewiesen oder erfasst wird.
  16. Verfahren nach Anspruch 15, wobei das Verfahren in Zellen durchgeführt wird.
  17. Verfahren nach einem der Ansprüche 15 bis 16, wobei das genannte Polypeptid einen Chromophor umfasst.
  18. Kit mit Teilen, die zur Durchführung eines Verfahrens nach einem der Ansprüche 16 bis 17 nützlich sind, der ein Polypeptid nach Anspruch 1 umfasst.
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